Structural determinants of human APOBEC3Aenzymatic and nucleic acid binding propertiesMithun Mitra1 Kamil Hercık1 In-Ja L Byeon23 Jinwoo Ahn23 Shawn Hill4

Kathyrn Hinchee-Rodriguez1 Dustin Singer1 Chang-Hyeock Byeon23

Lisa M Charlton23 Gabriel Nam1 Gisela Heidecker4 Angela M Gronenborn23 and

Judith G Levin1

1Section on Viral Gene Regulation Program on Genomics of Differentiation Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development National Institutes of Health Bethesda MD 20892-2780USA 2Department of Structural Biology University of Pittsburgh School of Medicine Pittsburgh PA 15261USA 3Pittsburgh Center for HIV Protein Interactions University of Pittsburgh School of Medicine PittsburghPA 15261 USA and 4HIV Drug Resistance Program Frederick National Laboratory for Cancer ResearchNational Cancer Institute Frederick MD 21702 USA

Received July 5 2013 Revised and Accepted September 25 2013

ABSTRACT

Human APOBEC3A (A3A) is a single-domain cytidinedeaminase that converts deoxycytidine residues todeoxyuridine in single-stranded DNA (ssDNA) Itinhibits a wide range of viruses and endogenousretroelements such as LINE-1 but it can also editgenomic DNA which may play a role in carcinogen-esis Here we extend our recent findings on theNMR structure of A3A and report structural bio-chemical and cell-based mutagenesis studies tofurther characterize A3Arsquos deaminase and nucleicacid binding activities We find that A3A bindsssRNA but the RNA and DNA binding interfacesdiffer and no deamination of ssRNA is detectedSurprisingly with only one exception (G105A)alanine substitution mutants with changes inresidues affected by specific ssDNA binding retaindeaminase activity Furthermore A3A binds anddeaminates ssDNA in a length-dependent mannerUsing catalytically active and inactive A3A mutantswe show that the determinants of A3A deaminaseactivity and anti-LINE-1 activity are not the sameFinally we demonstrate A3Arsquos potential to mutategenomic DNA during transient strand separationand show that this process could be counteractedby ssDNA binding proteins Taken together ourstudies provide new insights into the molecularproperties of A3A and its role in multiple cellularand antiviral functions

INTRODUCTION

The human APOBEC3 (A3) proteins are host restrictionfactors that exhibit activity against retroviruses and en-dogenous retroelements and play an important role inthe innate immune response to human pathogens (1ndash4)These proteins function as deoxycytidine deaminases thatconvert dC to dU in single-stranded DNA (ssDNA) andbehave as DNA mutators (56) The A3 family consists ofseven members having either one (A3A A3C and A3H)or two (A3B A3D A3F and A3G) zinc (Zn)-bindingdomains with the conserved motif HX1EX23ndash24CX2-4C

(X is any amino acid) (127) The cysteine and histidineresidues coordinate a zinc metal ion (Zn2+) and theglutamic acid is implicated in proton transfer during ca-talysis (18) The deamination target site specificity of A3proteins is determined by the nucleotides (nt) flanking dCresidues in the substrate (69)A3A is a single-domain deaminase that acts on the

cytosine bases in TC-containing ssDNA (10ndash16)moreover it can also deaminate methyl cytosine residues(17ndash19) It is expressed in peripheral blood mononuclearcells specifically in the cells of the CD14+ lineage thatincludes monocytes and macrophages (101220ndash22) Inaddition A3A is also expressed in psoriatic keratinocytesand in normal keratinocytes but only on treatment withan activator of protein kinase C (2324) Although A3Adisplays multiple biological activities deamination is notnecessarily required in each case Early studies showedthat A3A has robust activity against long interspersedelement-1 (LINE-1) and Alu retrotransposition in cell-based assays (101325ndash30) (reviewed in refs 3132)

To whom correspondence should be addressed Tel +1 301 496 1970 Fax +1 301 496 0243 Email levinjumailnihgovCorrespondence can also be addressed to Angela M Gronenborn Tel +1 412 648 9959 Fax +1 412 648 9008 Email amg100pittedu

Published online 24 October 2013 Nucleic Acids Research 2014 Vol 42 No 2 1095ndash1110doi101093nargkt945

Published by Oxford University Press 2013 This work is written by US Government employees and is in the public domain in the US

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Surprisingly no DNA mutations that could be ascribed toA3Arsquos deaminase activity were detected in LINE-1 andother retrotransposon DNA sequences suggesting that al-ternative mechanisms independent of A3Arsquos catalyticactivity might be involved (102627) A deaminase-inde-pendent mechanism was also indicated for A3A-mediatedinhibition of parvovirus replication (1033) HoweverA3Arsquos editing function was found to be required for itsability to mutate human papilloma virus DNA (34) todegrade foreign DNA in human cells (1215) and toblock replication of Rous sarcoma virus (35) and humanT-lymphotropic virus type 1 (HTLV-1) (36)Initially it was reported that A3A does not inhibit

HIV-1 infectivity in single-cycle and replication assays inT cells and other established cell lines (10112837ndash39)However several groups have provided evidence thatA3A inhibits HIV-1 replication in cells of myeloid origin(eg monocyte-derived macrophages) (204041) where itsexpression is stimulated by addition of interferon-alpha (1020ndash2242) Moreover A3A-specific editingof HIV-1 minus-strand DNA during reverse transcriptionwas found to occur in infected myeloid cells (40)suggesting a role for A3A deaminase activity in HIV-1restrictionA3A appears to play an important role as a cellular

defense protein but its DNA mutator activity could alsobe detrimental to the cell in which it is expressedMoreover as A3A localizes to the cytoplasm and to thenucleus in certain cell lines it has access to genomic DNA(1026273843) Studies using cell-based assays showedthat A3A has the potential to mutate genomic and mito-chondrial DNA (154445) In addition cellular expressionof A3A was shown to induce strand breaks trigger theDNA damage response and cause cell cycle arrest (4446)Thus it seems likely that cellular regulatory mechanismsmay be in place to modulate A3A function and preventthese harmful effects The human TRIB3 protein wasshown to lower the levels of genomic DNA editing byA3A (47) More recently however it was reported thatin myeloid cells A3A is found exclusively in the cyto-plasm suggesting that cytoplasmic retention in thesecells prevents damage to nuclear DNA (48)To elucidate the molecular mechanisms that govern the

biological functions of A3A we recently solved the A3Asolution structure at high resolution using NMR spectros-copy (16) The overall structure of A3A was found to besimilar to structures described for A3C (49) and for theC-terminal domain of A3G (A3G-CD2) (50ndash54) (reviewedin ref 55) despite some differences mainly in the loopregions We also defined the surface for A3A interactionwith short ssDNA substrates (15 nt) and performed adetailed analysis of substrate binding and specificityOur results showed that the binding and deaminationactivity of A3A with ssDNA oligonucleotides containingTTCA and ACCCA motifs are similar consistent with theA3A editing site preference in cell-based assays ie pre-dominantly in TC- and CC-containing regions(12153436)Here we report the biochemical and biological

properties of A3A taking advantage of the structural in-formation in our earlier study (16) Initially we probed the

interaction of A3A with a 9-nt ssRNA by NMR Incontrast to previous results obtained with a 9-ntssDNA of the same sequence (16) we found thatssRNA is not a substrate for A3A deaminase activityand is bound more weakly to the enzyme Using struc-ture-guided mutagenesis point mutations wereintroduced into residues identified as important forspecific binding to ssDNA substrates and the effect ondeaminase activity was measured In addition longerssDNAs (20 nt) were used to determine the oligonucleo-tide length dependence for ssDNA deamination andnucleic acid binding Finally we explored the role ofdeaminase and nucleic acid binding properties of A3Ain relation to its biological activities ie inhibition ofLINE-1 retrotransposition and HIV-1 reverse transcrip-tion as well as genomic DNA editing which can lead totumor formation

MATERIALS AND METHODS

Materials

Unlabeled DNA oligonucleotides used for electrophoreticmobility shift assays (EMSA) deaminase assays andcloning were obtained from Lofstrand Labs Ltd(Gaithersburg MD USA) All RNA oligonucleotidesand DNA oligonucleotides labeled with AlexaFluor488 were purchased from Integrated DNATechnologies (Coralville IA USA) Oligonucleotidesused for deaminase assays and EMSA were eithergel-purifed or were subjected to purification using high-pressure liquid chromatography (HPLC) and are listed inTable 1 Oligonucleotide concentrations were determinedby absorbance at 260 nm using the extinction coefficientprovided by the manufacturer [g-33P]ATP and [g-32P]ATP(each 3000Cimmol) were purchased from PerkinElmer(Waltham MA USA) HIV-1 reverse transcriptase (RT)was obtained from Worthington Biochemical Corp(Lakewood NJ USA) T4 polynucleotide kinase andEscherichia coli (E coli) uracil DNA glycosylase (UDG)were purchased from New England Biolabs (Beverly MAUSA) Nuclease-free water SUPERaseIn Proteinase Kand gel loading buffer were obtained from Ambion

(Life Technnologies Corp Grand Island NY USA)Rabbit anti-A3G antibody (ApoC17) (56) a highlycross-reactive antiserum used for detection of A3Aproteins was a generous gift from Dr Klaus Strebel(NIAID NIH) Anti-tubulin antibody was obtainedfrom Abcam (Cambridge MA USA) HIV-1 nucleocap-sid protein (NC) was kindly provided by Dr Robert JGorelick (SAIC-Frederick Inc) T4 Gene 32 and E coliSSB proteins were purchased from Affymetrix Inc (SantaClara CA USA)

A3A expression in E coli and purification

Synthetic A3A genes with a C-terminal His6-tag(LEHHHHHH) were inserted into the NdeIndashXhoI siteof the pET21 plasmid (Novagen) for expression inE coli Rosetta 2 (DE3) and purified as described previ-ously (16) Recombinant proteins were estimated to begt95 pure as determined by SDS-polyacrylamide gel

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electrophoresis (SDS-PAGE) and 2D 1H-15Nheteronuclear single quantum coherence (HSQC) spec-troscopy (16) The protein concentration was determinedusing the BCA protein assay kit (Pierce Thermo FisherScientific Inc Rockland IL USA) and verified by aminoacid analysis of an acid hydrolysate (Keck BiotechnologyResource Laboratory Yale University New Haven CTUSA) To check for nuclease contamination in each A3Apreparation different amounts of A3A protein wereincubated with 32P-labeled DNA or RNA oligonucleo-tides Analysis by PAGE indicated that the A3A prepar-ations were virtually nuclease-free (data not shown)The E72Q Y130F and D131E mutant proteins wereconstructed expressed and purified as described above(16) These proteins were shown to exhibit a wild-type(WT) fold by 1H-15N HSQC NMR spectroscopy(Supplementary Figure S1)

Real-time NMR studies of ssRNA deamination

A3A (final concentration 007mM) was added to a HPLC-purified 9-nt ssRNA oligonucleotide (112mM)AUUUCAUUU (JL1181 Table 1) in 25mM sodiumphosphate buffer (pH 69) containing 10mM DTTDeamination was monitored by acquiring a time seriesof 2D 1H-13C HSQC spectra at 900MHz 25C (16)

Mapping the A3A ssRNA binding site and titrationby NMR

To monitor ssRNA binding to A3A aliquots of a 20mMAUUUCAUUU stock solution were added to 0074mM13C15N-labeled A3A in 25mM sodium phosphate buffer(pH 69) containing 10mM DTT A series of 2D 1H-15NHSQC spectra were acquired at 900MHz 25C

1H15N-combined chemical shift changes were calculated

usingffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2HN+ethN=6THORN

2q

with HN and N the1HN

and 15N chemical shift differences observed for A3Abefore and after adding nucleotides (90 saturation)The residues showing large (gt005 ppm)1H15N-combined chemical shift changes were mappedon the A3A NMR structure (16) Binding isothermswere obtained by plotting 1HN proton chemical shiftchanges observed for F54 W98 S103 and W104 versusnucleotide concentration Dissociation constants (Kd)were calculated by non-linear best fit of the data usingKaleidaGraph (Synergy Software Reading PA USA)with subsequent averaging of the Kd values of all fouramino acid resonances All of the structure figures weregenerated using MOLMOL (57)

Construction of A3A mutants for cell-based assays

The A3A coding sequence (Genbank Accession NM_145699) was inserted into a pCDNA 31() vector(Invitrogen Life Technologies Corp) for expression inmammalian cells All of the A3A mutants were con-structed using the QuikChange Lightning Site-DirectedMutagenesis kit (Agilent Technologies Santa Clara CAUSA) The primers for mutant construction were designedusing the online QuikChange Primer Design Programprovided by the manufacturer Large-scale plasmid prep-aration was performed using the HiSpeed Plasmid MaxiPrep kit (Qiagen Inc Germantown MD USA) For eachplasmid the sequence of the insert was verified by DNAsequencing performed by ACGT Inc (Wheeling ILUSA)

Table 1 List of oligonucleotides used for EMSA and deaminase assays

Name Purpose Length(nt)

Sequencea

JL-895 EMSA Deamination 40 50-ATT ATT ATT ATT ATT ATT ATT TCA TTT ATT TAT TTA TTT A-30

JL-913 Deamination 40 50-(Alexa488) ATT ATT ATT ATT ATT ATT ATT TCA TTT ATT TAT TTA TTT A-30

JL-935 EMSA Deamination 40 50-ATT ATT ATT ATT ATT ATT ATT TUA TTT ATT TAT TTA TTT A-30

JL-974 EMSA 30 50-TTA TTA TTA TTA TTT CAT TTA TTT ATT TAT-30

JL-975 EMSA 20 50-ATT ATT ATT TCA TTT ATT TA-30

JL-984 EMSA Deamination 40 50-TAA ATA AAT AAA TAA ATG AAA TAA TAA TAA TAA TAA TAA T-30

JL-986 EMSA Deamination 60 50-ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT TCA TTT ATT TAT TTA TTTATT TAT TTA-30

JL-987 EMSA 70 50-ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT TCA TTT ATT TATTTA TTT ATT TAT TTA TTT A-30

JL-988 EMSA Deamination 80 50-ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT TCA TTTATT TAT TTA TTT ATT TAT TTA TTT ATT TA-30

JL1152 Deamination 20 50-(Alexa488) ATT ATT ATT TCA TTT ATT TA-30

JL1153 Deamination 60 50-(Alexa488) ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT TCA TTT ATT TATTTA TTT ATT TAT TTA-30

JL-1045 EMSA 40 50-auu auu auu auu auu auu auu uca uuu auu uau uua uuu a-30

JL-970 EMSA 40 50-uaa aua aau aaa uaa aug aaa uaa uaa uaa uaa uaa uaa u-30

JL1181 Deamination 9 50-auu uca uuu-30

JL-1088 Deamination 40 50-TAA ATA AAT AAA TAA ATC AAA TAA TAA TAA TAA TAA TAA T-30

JL-1089 Deamination 40 50-TAA ATA AAT AAA TAA AAC TAA TAA TAA TAA TAA TAA TAA T-30

JL-1090 Deamination 40 50-TAA ATA AAT AAA TAA TAC TTA TAA TAA TAA TAA TAA TAA T-30

JL-1091 Deamination 40 50-TAA ATA AAT AAA TTT TAC TTT AAA TAA TAA TAA TAA TAA T-30

JL-1095 Deamination 40 50-(Alexa488) ATT ATT ATT ATT ATT ATT ATA CCC AAA ATT TAT TTA TTT A-30

aUpper and lower case letters represent DNA and RNA oligonucleotides respectively

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Cell culture transfection procedure and preparation ofmammalian cell extracts

The 293T cells were propagated in Dulbeccorsquos modifiedEaglersquos medium (Invitrogen) containing 10 fetalbovine serum (Hyclone now Thermo Scientific) 2mMglutamine penicillin (50 IUml) and streptomycin(50mgml) The cells (24 105well in a 12-well plate)were transfected with 075 mg of either empty pCDNA31() vector or an A3A-containing plasmid (WT ormutant) using GenJetTM In Vitro DNA TransfectionReagent (SignaGen Laboratories Gaithersburg MDUSA) according to the manufacturerrsquos instructionsFollowing incubation for 48 h at 37C in an incubatorwith 5 CO2 the cells were harvested and lysed usingM-PER mammalian protein extraction reagent (ThermoFisher Scientific Inc Rockford IL USA) as described inthe manufacturerrsquos manual in the presence of CompleteProtease Inhibitor Cocktail (Roche Diagnostics CorpIndianapolis IN USA) After cell lysis NaCl (final con-centration 100mM) and glycerol (final concentration10 volvol) were added and the resulting mixture wascentrifuged at 13 000 g for 10min The total concentrationof protein in the supernatant was determined using theBCA protein assay kit To check A3A expression levelsin each sample 20 mg of total protein was subjected towestern blot analysis using the Western Breeze chemilu-minescent Western blot immunodetection kit (Invitrogen)and either anti-A3G (for A3A expression) or anti-tubulin(loading control) as primary antibodies

Deaminase assays

Deaminase assays were performed using a protocoladapted from the procedure described by Iwatani et al(58) The substrate was a 40-nt Alexa-Fluor 488-labeledTTCA-containing ssDNA (JL913) (Table 1) unlessspecified otherwise Activity of cell extracts was evaluatedin reactions (final volume 40 ml) containing the ssDNAsubstrate (final concentration 180 nM) reaction buffer[10mM TrisndashHCl buffer (pH 80) 50mM NaCl 1mMdithiothreitol (DTT) 1mM EDTA and 4U of UDG]and increasing amounts of extract (total protein expressedin mg) as specified incubation was for 1 h at 37CReactions were terminated by further incubation at 37Cwith 10 ml of 1N NaOH for 15min followed by neutral-ization with 10 ml of 1N HCl Analysis was performed byelectrophoresis on a 10 denaturing polyacrylamide gelas detailed in Byeon et al (16) Gels were scanned in fluor-escence mode on a Typhoon 9400 Imager (GE Healthcare)and the data were quantified using ImageQuant softwareThe values for percent deaminase product formed in eachreaction were calculated by dividing the product signalintensity by the total signal intensity in the lane and multi-plying by 100 The average of two independent experi-ments is shown in the figuresDeaminase activity of purified A3A using a UDG-

dependent gel-based assay was measured as described pre-viously (16) The data are the average values from threeindependent experiments The catalytic C in TTCA andCCCA motifs is underlined throughout the text

EMSA

EMSA assays were performed with ss and double-stranded (ds) oligonucleotides (Table 1) using theprotocol provided in Byeon et al (16) Radioactivity wasquantified by using the Typhoon 9400 Imager andImageQuant software The values for percent A3A-bound nucleic acid were calculated by dividing the inten-sity of the shifted band (A3A-bound nucleic acid) by thetotal intensity in the lane (the sum of the intensities of theshifted band the free nucleic acid band and the areabetween the shifted and free nucleic acid bands) The A3A-bound nucleic acid values of at least three independ-ent experiments were averaged (standard deviation5ndash10)

Retrotransposition assay

The assay uses a vector that is similar to the replication-dependent LINE-1 vector originally designed by DGarfinkel T Heidmann and J Moran (59ndash61) Theplasmid used in this study was constructed as follows (i)a firefly luciferase (luc) expression cassette driven by acytomegalovirus immediate early promoter wasembedded in the 30UTR of the LINE-1 transcriptionunit such that the luc cassette and LINE-1 are transcribedin opposite orientations and (ii) a rabbit g-globin intronwas inserted into the luc sequence in the sense orientationof the LINE-1 transcript thereby disrupting the luc openreading frame on the plasmid construct and preventingexpression of luciferase from the transfected plasmidAfter transfection into 293T cells the LINE-1 sequenceis transcribed the intron is removed by splicing and thetranscript is reverse transcribed and integrated As a con-sequence the now uninterrupted luc gene directs the syn-thesis of firefly luciferase whose activity serves as ameasure of the retrotransposition events that occurred

For the retrotransposition assay a constant amount ofthe LINE-1 plasmid (075 mg) was transfected into 293Tcells (see above) with 01 or 05 mg of pCDNA 31()plasmids encoding A3A WT or mutant proteins in 12-well plates The total amount of pCDNA 31() plasmidwas set at 075mg for each transfection by adding either065 or 025mg of empty vector respectively For controlswithout A3A 075 mg of empty vector was used Threedays after transfection the cells were lysed and luciferaseactivity was determined using the Bright-GloTM luciferaseassay system (Promega Corp Madison WI USA) ac-cording to the manufacturerrsquos instructions 100luciferase activity corresponds to no inhibition whereas0 activity signifies complete inhibition of retrotran-sposition Results for retrotransposition assays shown inthe figures represent the average of two or three independ-ent experiments

HIV-1 minus-strand transfer assay

Assay of the minus-strand transfer step in reverse tran-scription (62ndash64) was performed using the reaction condi-tions described previously (6566) except that HIV-1 NCwas not added Briefly acceptor RNA (148 nt) was heatannealed to 33P-labeled () strong-stop DNA [()

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SSDNA] (128 nt) in buffer containing 50mM TrisndashHCl(pH 80) and 75mM KCl The annealed duplex containsa 54-nt 50 ssRNA overhang which serves as the templatefor extension of the annealed () SSDNA [see diagramin Figure 1C in (66)] Varying amounts of purified A3A(WT or E72Q mutant) as specified were added and themixture was incubated at 37C for 10min after whichHIV-1 RT dNTPs and MgCl2 (final concentration1mM) were added with further incubation at 37C for1 h Termination of the reactions electrophoresis indenaturing gels and PhosphorImager analysis were per-formed as described (67)

RESULTS

NMR analysis to assess ssRNA deamination and bindingto A3A

Human A3 proteins are thought to use ssDNA as theexclusive substrate for deaminase activity The inabilityto act on ssRNA has been shown experimentally forpurified A3G (58) and in early work Madsen et al (24)reported that A3A (known as phorbolin-1 at that time)could not deaminate a mononucleoside or apolipoprotein

B cRNA In our recent study of the A3A NMR solutionstructure (16) (Figure 1) we followed deamination ofssDNA using a real-time NMR assay Here we adopteda similar approach to determine whether A3Acan deaminate a 9-nt ssRNA with the sequenceAUUUCAUUU (Table 1 JL1181) (Figure 2A) The in-tensity of the 1H-13C HSQC resonance from the onlycytosine in the ssRNA sample (112mM) which resonatesat 5945 ppm (1H) and 9884 ppm (13C) did notdecrease after addition of A3A (007mM) and incubationfor 15 h (left) and 96 h (right) This result is in starkcontrast to our previous observation with the correspond-ing 9-nt ssDNA ATTTCATTT which was completelydeaminated within 1min under similar conditions (16)Although unlikely secondary structure formation couldrender the cytosine inaccessible to the enzyme weevaluated this possibility by 1H 1D NMR No imino res-onances compatible with base pairing in the RNA oligo-nucleotide were detected confirming the ss nature of theRNA

1H-15N HSQC spectra were used to study binding of the9-nt ssRNA to A3A Figure 2B shows titration isothermsobtained by monitoring changes of amide resonances forseveral representative A3A residues that are perturbed by

A3AA3G-CTD

A3AA3G-CTD

A3AA3G-CTD

67

137

199384

254

322

Loop 7

E138L135

W104 G105G65F66

C106

C101E72

R69F102

Y130D131Y132P134

I26

R191

loop 7

A

B C

Figure 1 (A) Sequence alignment of A3A (residues 1ndash199) with A3G-CD2 (residues 194ndash384) The Zn-binding residues and the catalytic glutamic acidresidue are highlighted in blue and the loop 7 region (residues 127ndash135) of A3A is marked The A3A residues mutated in this study are labeled in greenasterisks The sequence alignment was performed using Lasergene software (DNASTAR Inc Madison WI USA) (B) and (C) A3A ribbon structureswith residues that were mutated and tested (Figures 3B 5 and 6C) highlighted in green The coordinated Zn2+ ion is shown as a brown ball

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the ssRNA binding Similar titration isotherms wereobserved for these resonances yielding a Kd value of042plusmn007mM This indicates that RNA binding isspecific albeit approximately one order of magnitudeweaker than binding of the corresponding 9-nt DNA(Kd=006plusmn001mM) (16) In gel-shift assays with a40-nt ssRNA the binding efficiency appears to be3-fold higher for ssRNA than for the corresponding40-nt ssDNA (16) This result is most likely caused byboth specific and non-specific binding to a long RNA aswell as differences in the EMSA and NMR conditions(eg differences in oligonucleotide length)The RNA binding site (Figure 2C) was mapped onto

the A3A NMR structure (PDB code 2m65) and comparedwith the previously determined DNA binding site(Figure 2D) (16) Although the binding interfaces aresimilar interesting differences were observed Clearly

the number of residues that are located on the bindingsurface when A3A is bound to RNA is smaller thanwhen it is bound to DNA Most noticeably the K30N57 and Q58 amide resonances exhibited little perturb-ation on RNA binding whereas they experienced largechanges on DNA binding This is significant as theseamino acids reside at the bottom of the deep bindingpocket that accommodates the reactive cytosine base inssDNA (16) Thus the data suggest that a cytosine ringin an RNA substrate cannot be positioned accurately inthe catalytic site for deamination

Differential role of A3A amino acids in nucleic acidbinding and deaminase activity

The NMR study allowed us to identify A3A residues thatare affected by specific binding of short ssDNA substratescontaining a single TTCA deaminase recognition site (16)

5860

100

102

104

106

5860

98

56 56

C C

U-3U-2U-1U+2U+3U+4

1H (ppm) 1H (ppm)

13C

(pp

m)

15 h 96 h

06-008-006-004-002000002004006008010012

00 02 04 0806 10 12

F54 1HN

W98 1Hε

S103 1HN

W104 1Hε1 H ch

emica

l shif

t cha

nge (

ppm

)

[rAUUUCAUUU] (mM)

B

C

A

rAUUUCAUUU binding site dATTTCATTT binding site

Kd = 042 plusmn 007 mMU-3U-2U-1U+2U+3U+4

L66W104

Y132D133

E138S103 H70

H16I17

G105

K30T31Y32

N57Q58 K60

C64G65L66

L63

W104

F102 H70

Y132D133D131

E138L135

K137Y136

S103C101

G105

I17

D

Figure 2 Deamination and binding assays with A3A and a 9-nt ssRNA (A) Deamination of the single cytosine in the ssRNA oligonucleotideAUUUCAUUU (JL1181 Table 1) was monitored by following the 13C-5-1H resonances of cytosine and uracil in 2D 1H-13C HSQC spectra Thetotal reaction time is indicated at the top right Binding isotherms for representative HN resonances (B) and mapping of the binding site onto theA3A structure (C) for ssRNA (AUUUCAUUU) interacting with A3A monitored by 1H-15N HSQC spectroscopy (D) Binding site of ssDNAATTTCATTT mapped onto the A3A structure (16) for comparison In (C) and (D) A3A residues whose resonances exhibit large 1H15N-combinedchemical shift changes on nucleotide addition are colored red (gt 005 ppm) and orange (0028ndash0050 ppm)

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These residues include amino acids that may play a directrole in deamination and those that may interact with theneighboring nucleotides on both sides of the reactive dC(TTCA) Non-catalytic residues that exhibit the largestchemical shift perturbations include W104 G105 L135and E138 The two-residue WG sequence is unique toA3A and the C-terminal domain of A3B In additionthe G65 and F66 residues located at the center of aflexible loop (loop 3 residues 57ndash70) that undergoes con-formational exchange in free A3A also show largechemical shift perturbations on binding of ssDNA sub-strates (16)

To determine whether any of these residues (Figure 1Aand B) are important for A3A catalytic function we per-formed alanine scanning mutagenesis and tested themutant cell lysates in deaminase assays (Figure 3) Allmutant proteins except G105A exhibited deaminaseactivity albeit to varying degrees (Figure 3A) Theprotein expression levels of all variants were similar(Figure 3B) G105A lacked activity even when highamounts of protein were present in the reaction Theseresults suggest that although all of the residues areaffected by specific binding to ssDNA substrates not sur-prisingly the individual contributions to A3Arsquos deaminaseactivity are not equivalent Data on other A3A residuesthat interact with nucleic acids are presented below

A3A binds and deaminates ssDNA oligonucleotides in alength-dependent manner

As we previously mapped the nucleic acid binding sitesfor short (15 nt) ssDNA substrates (16) it was ofinterest to further investigate A3Arsquos binding propertiesby using longer ss and ds nucleic acids (20 nt) Initiallywe determined whether A3A binds to ssDNA in a length-dependent manner (Figure 4A) Gel analysis (EMSA)showed that the amount of bound ssDNA increasedwith increasing oligonucleotide length from 20 to80 nt For oligonucleotides 60 nt at the highest proteinconcentration used (100mM) 73ndash82 of the input DNAwas complexed to protein Like A3G (5868) A3Ainteracts poorly with ds nucleic acids with a rankorder of binding of dsDNAgtDNA-RNAdsRNA(Supplementary Figure S2)

To determine whether substrate length influencesenzymatic activity A3A was incubated with 20- 40- and60-nt ssDNA substrates (Figure 4B) There was no signifi-cant difference in the amount of deaminase product when200 nM A3A was present in the reaction However with100 nM A3A activity was highest with the 20-nt ssDNAand was reduced by 20 and 25 when the 40- and 60-nt substrates were used respectively presumably becausebinding to the larger substrates was not saturated with thelower amount of A3A Interestingly in NMR experimentswith ssDNAs that were 6 9 or 15 nt in length theapparent second-order rate constants (kcatKM) for thedeaminase activity of the 9- and 15-nt substrates wereidentical Moreover these rate constants were 3-foldhigher than the value obtained with the 6-mer (16)mainly due to the tighter binding of the larger ssDNAs(smaller KM) This suggests that the minimum substrate

length for optimal deaminase activity is between 6 and9 nt

Relationship between A3A deaminase activity andinhibition of LINE-1 retrotransposition

An important aspect of this study is to relate our findingson A3Arsquos binding and deaminase properties to its biolo-gical functions We first consider this issue in regard toLINE-1 retrotransposition Although A3A has beenreported to be a potent inhibitor of LINE-1 retrotran-sposition in cell-based assays (reviewed in refs 3132)the underlying mechanism is still unclear and questionsremain as to whether A3A deaminase activity is necessaryfor this activity To investigate this issue we changed A3Aresidues that might be important for deamination andorinhibition of LINE-1 activity A3A residues that weremutated are highlighted with green asterisks inFigure 1A and are marked on the A3A NMR structurein Figure 1C Deaminase assays were performed withextracts from 293T cells that were transfected withvectors carrying the A3A genes (WT or mutants) Theamounts of expressed protein for WT and all of theA3A mutants were similar (Figures 5C and 6C and F)No A3A was present in cells with the empty vector(Figure 5C) and the extract was therefore devoid ofdeaminase activity (Figure 6D)As controls the activities of A3A variants with changes

in the highly conserved active site cysteine and glutamicacid residues were tested as well (Figure 5) Substitution ofcysteines 101 and 106 by serine (C101S and C106S) whichabolishes Zn binding led to complete loss of deaminaseactivity as expected (Figure 5A) Similarly substitution ofglutamic acid 72 by aspartic acid (E72D) or glutamine(E72Q) also resulted in abrogation of deaminase activity

0

20

60

80

D

eam

inas

e P

rod

uct

40

100A

B

0 1 2 3 4 5

WTW104AE138A

L135A

F66A

Total Protein (microg)

G65A G105A

Empt

y W

TW

104A

E138A

L135

AF6

6A

G65

A

G10

5A

A3A

Tubulin

Figure 3 Deaminase activity of A3A mutants Residues were selectedfor mutagenesis based on our earlier NMR results (16) (A) Deaminaseactivity (B) Expression of A3A WT and mutants in 293T cells detectedby western blot analysis as described in lsquoMaterials and Methodsrsquo

Nucleic Acids Research 2014 Vol 42 No 2 1101

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

80 n

t

1

2

3

4

5

6

7

8

9

10

2

16

55

72

82

2

7

46

64

74

no

no

70 n

t 60 n

t

11

12

13

14

15

1

6

17

18

1

9

2

0

1

4

4

4

6

4

7

3

2

4

20

33

39

no

no

40 n

t 30 n

t

2

1 22 23 24 2

5 26 27 28 2

9 30

Fre

e D

NA

0

1

2

6

12

0

1

1

2

3

S

hif

ted

(

)

no

A3A

no

20 n

t A B

0206080 40100

120

Deaminase Product

100

nM

A3A

200

nM

A3A

A

3A-B

ou

nd

D

NA

1 2 3 4

5

Fre

e D

NA

WT

E72Q

2

9

4

95

8

0

83

90

9

1

9

3

94

91

92

1

Sh

ifte

d (

)

6

7 8 9 1

0 11 12

WT

Y130F

D131E

C

A

3A-B

ou

nd

D

NA

no

no

A3A

Figure

4A3A

EMSA

anddeaminase

assayswithssDNAsofdifferentlengths

(A)BindingofA3A

toTTCA-containingDNA

oligonucleotides

ofvaryinglengths(20nM)(Table

1)

80nt

(JL988)70nt(JL987)60nt(JL986)40nt(JL895)30nt(JL974)and20nt(JL975)Lanes1611162126noA3A271217222725mM381318232850mM4914192429

75mM51015202530100mMThepercentageofoligonucleotideboundto

A3A

isindicatedatthebottom

ofthegel

imageaspercentshiftedThepositionsoffree

oligonucleotideand

shiftedbands(A

3A-boundnucleicacid)are

alsoindicatedThecalculationswereperform

edasdescribed

inlsquoM

aterials

andMethodsrsquo(B

)Deaminase

assaysusing100nM

and200nM

purified

WT

A3A

andssDNAswithdifferentlengths(180nM)(Table

1)

JL1152(20nt

whitebar)JL

913(40ntblack

bar)

andJL

1153(60nt

graybar)(C

)Bindingofan80-ntTTCA-containing

ssDNA

(JL988)by60mM

(lanes

2468and10)or80mM

(lanes

3579and11)A3ALanes1and12noA3A236and7WTA3A4and5E72Q8and9Y130F10and11D131E

Thepositionsoffree

andprotein-boundDNA

andthepercentshiftedare

indicated

1102 Nucleic Acids Research 2014 Vol 42 No 2

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

(Figure 5A) These results are in accord with previousmutational studies of active site residues (1013)

Whether A3A deaminase activity is necessary for theinhibition of LINE-1 retrotransposition was also testedusing these mutants (Figure 5B) As expected WT A3Adisplayed potent anti-retrotransposition activity (2luciferase activity) in agreement with earlier studies(101325ndash30) The E72Q and C101S mutants had essen-tially no inhibitory activity in contrast the C106S mutantexhibited moderate anti-LINE-1 activity whereas theE72D mutant strongly inhibited retrotransposition Thebehavior of the E72D mutant contrasts with the lack ofinhibitory activity displayed by other Glu mutants E72Q(Figure 5B) (10) and E72A (1327)

Deaminase and retrotransposition activities of non-active site mutants including those in loop regions(Figure 1A and C) were also determined (Figure 6)Although A3 loop residues do not directly participate incatalysis loop 7 residues in A3G-CD2 were shown to playa role in the preferential deamination of CCC-containing

ssDNA (6516970) Consistent with these observationsmutation of A3A loop 7 residues (Y130 D131 Y132)either singly or in pairs completely abolished deaminaseactivity (Figure 6A) Moreover exchanging the entireA3A loop 7 sequence for that of A3G-CD2 (Figure 1A)(lsquoLoop 7rsquo mutant) caused complete loss of deaminaseactivity (Figure 6A) Interestingly although P134 is partof a surface that interacts with the 50 T in the TTCA rec-ognition motif the P134A loop 7 mutation had only asmall effect on deamination (Figure 6D) In addition toP134A other non-catalytic site mutants (R29A I26GR191D and F102) also retained activity with ssDNA sub-strates albeit at different levels (Figure 6D) This behaviorreflects their different locations in the A3A protein struc-ture which could influence the mode of interaction withnucleic acidsWith the exception of D131E which displayed a high

level of anti-LINE-1 activity all of the other loop 7mutants (Figure 6A) were able to only weakly inhibitretrotransposition (Figure 6B) The dual loss of activityfor most loop 7 mutants may be related to the possible lossof the structural integrity of the mutant proteins Severaldeaminase-active mutants displayed anti-retrotran-sposition activities that were as high as that of WT(F102A and P134A Figure 6E) or had substantial inhibi-tory activity (R69A I26G and R191D Figure 6E)However taken together the data in Figures 5 and 6indicate that in most cases total loss of deaminaseactivity is not necessarily associated with an inability toinhibit LINE-1 retrotranspositionPrevious studies of A3A inhibition of retrotransposition

(see above) suggested that a deaminase-independent mech-anism might be involved To further investigate this hy-pothesis we performed retrotransposition assays usingdifferent amounts of WT and mutant A3A plasmidsInterestingly the inhibitory activity increased when theamount of transfected DNA was increased (01 and05mg) The dose dependence was particularly strikingfor the E72D (Figure 5B) D131E and Y130F (Figure6B) mutants that displayed no deaminase activity evenat high total protein concentrations (Figure 5A and 6Arespectively) This gain-in-function (enhanced anti-retrotransposition activity) for most of the deaminase-negative mutants suggests the possible existence of add-itional mechanism(s) that do not require A3A catalyticactivityFor example deaminase-independent inhibition of

LINE-1 retrotransposition by A3A might involvebinding to ssDNA intermediate(s) If that were the casewe would expect that the level of anti-LINE-1 activitywould be related to the efficiency of ssDNA binding Toprobe this possibility we evaluated binding to an 80-ntssDNA (Figure 4C) using three A3A mutant proteinsE72Q Y130F and D131E which were all deaminase-negative (Figures 5A and 6A) but exhibiting differentlevels of anti-LINE-1 activity (Figures 5B and 6B)Surprisingly all of the mutants displayed binding levelssimilar to that of WT even though they had non-detect-able (E72Q) modest (Y130F) or strong (D131E) anti-LINE-1 activities Thus it would appear there is no

0

20

60

80

D

eam

inas

e P

rod

uct

40

100A

0

20

60

80

L

uci

fera

se A

ctiv

ity

40

100

B

0 1 2 3 4 5

Empty

WTC101SC106S

E72D

E72Q

Total Protein (microg)

120

140

WT C101S C106S E72D E72Q

C

A3A

Tubulin

Empt

y W

TC10

6SE72

DC10

1S

WT

E72Q

A3A

Tubulin

Figure 5 Deaminase and retrotransposition activities of A3A WT andactive site mutants (A) Deaminase assays using 293T cell extracts ex-pressing WT and active site mutants of A3A (B) Retrotranspositionassays to monitor the effect of active site mutations of A3A on LINE-1activity A description of the assays is given in lsquoMaterials andMethodsrsquo The gray and black bars represent transfection of 01 and05 mg of A3A WT or mutant plasmids respectively (C) Western blotanalysis of A3A WT and active site mutant expression in 293T cells

Nucleic Acids Research 2014 Vol 42 No 2 1103

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

(Figure 5A) These results are in accord with previousmutational studies of active site residues (1013)

Whether A3A deaminase activity is necessary for theinhibition of LINE-1 retrotransposition was also testedusing these mutants (Figure 5B) As expected WT A3Adisplayed potent anti-retrotransposition activity (2luciferase activity) in agreement with earlier studies(101325ndash30) The E72Q and C101S mutants had essen-tially no inhibitory activity in contrast the C106S mutantexhibited moderate anti-LINE-1 activity whereas theE72D mutant strongly inhibited retrotransposition Thebehavior of the E72D mutant contrasts with the lack ofinhibitory activity displayed by other Glu mutants E72Q(Figure 5B) (10) and E72A (1327)

Deaminase and retrotransposition activities of non-active site mutants including those in loop regions(Figure 1A and C) were also determined (Figure 6)Although A3 loop residues do not directly participate incatalysis loop 7 residues in A3G-CD2 were shown to playa role in the preferential deamination of CCC-containing

ssDNA (6516970) Consistent with these observationsmutation of A3A loop 7 residues (Y130 D131 Y132)either singly or in pairs completely abolished deaminaseactivity (Figure 6A) Moreover exchanging the entireA3A loop 7 sequence for that of A3G-CD2 (Figure 1A)(lsquoLoop 7rsquo mutant) caused complete loss of deaminaseactivity (Figure 6A) Interestingly although P134 is partof a surface that interacts with the 50 T in the TTCA rec-ognition motif the P134A loop 7 mutation had only asmall effect on deamination (Figure 6D) In addition toP134A other non-catalytic site mutants (R29A I26GR191D and F102) also retained activity with ssDNA sub-strates albeit at different levels (Figure 6D) This behaviorreflects their different locations in the A3A protein struc-ture which could influence the mode of interaction withnucleic acidsWith the exception of D131E which displayed a high

level of anti-LINE-1 activity all of the other loop 7mutants (Figure 6A) were able to only weakly inhibitretrotransposition (Figure 6B) The dual loss of activityfor most loop 7 mutants may be related to the possible lossof the structural integrity of the mutant proteins Severaldeaminase-active mutants displayed anti-retrotran-sposition activities that were as high as that of WT(F102A and P134A Figure 6E) or had substantial inhibi-tory activity (R69A I26G and R191D Figure 6E)However taken together the data in Figures 5 and 6indicate that in most cases total loss of deaminaseactivity is not necessarily associated with an inability toinhibit LINE-1 retrotranspositionPrevious studies of A3A inhibition of retrotransposition

(see above) suggested that a deaminase-independent mech-anism might be involved To further investigate this hy-pothesis we performed retrotransposition assays usingdifferent amounts of WT and mutant A3A plasmidsInterestingly the inhibitory activity increased when theamount of transfected DNA was increased (01 and05mg) The dose dependence was particularly strikingfor the E72D (Figure 5B) D131E and Y130F (Figure6B) mutants that displayed no deaminase activity evenat high total protein concentrations (Figure 5A and 6Arespectively) This gain-in-function (enhanced anti-retrotransposition activity) for most of the deaminase-negative mutants suggests the possible existence of add-itional mechanism(s) that do not require A3A catalyticactivityFor example deaminase-independent inhibition of

LINE-1 retrotransposition by A3A might involvebinding to ssDNA intermediate(s) If that were the casewe would expect that the level of anti-LINE-1 activitywould be related to the efficiency of ssDNA binding Toprobe this possibility we evaluated binding to an 80-ntssDNA (Figure 4C) using three A3A mutant proteinsE72Q Y130F and D131E which were all deaminase-negative (Figures 5A and 6A) but exhibiting differentlevels of anti-LINE-1 activity (Figures 5B and 6B)Surprisingly all of the mutants displayed binding levelssimilar to that of WT even though they had non-detect-able (E72Q) modest (Y130F) or strong (D131E) anti-LINE-1 activities Thus it would appear there is no

0

20

60

80

D

eam

inas

e P

rod

uct

40

100A

0

20

60

80

L

uci

fera

se A

ctiv

ity

40

100

B

0 1 2 3 4 5

Empty

WTC101SC106S

E72D

E72Q

Total Protein (microg)

120

140

WT C101S C106S E72D E72Q

C

A3A

Tubulin

Empt

y W

TC10

6SE72

DC10

1S

WT

E72Q

A3A

Tubulin

Figure 5 Deaminase and retrotransposition activities of A3A WT andactive site mutants (A) Deaminase assays using 293T cell extracts ex-pressing WT and active site mutants of A3A (B) Retrotranspositionassays to monitor the effect of active site mutations of A3A on LINE-1activity A description of the assays is given in lsquoMaterials andMethodsrsquo The gray and black bars represent transfection of 01 and05 mg of A3A WT or mutant plasmids respectively (C) Western blotanalysis of A3A WT and active site mutant expression in 293T cells

Nucleic Acids Research 2014 Vol 42 No 2 1103

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

direct correlation between ssDNA binding activity and theability to inhibit LINE-1 retrotransposition

A3A does not block nucleotide incorporation byHIV-1 RT

Another biological function of A3A is its ability to inhibitHIV-1 replication in myleoid cells (204041) A3A wasshown to inhibit synthesis of viral DNA during HIV-1reverse transcription and it was suggested that thisactivity depends on A3Arsquos ability to edit viral ssDNA(40) In the case of A3G in vitro assays (71ndash74) as wellas assays in virus-infected cells (75ndash79) provided evidencethat A3G can inhibit reverse transcription by deaminase-dependent and deaminase-independent mechanismsBased on A3Grsquos biochemical properties ie its slowdissociation from bound nucleic acid (7174) and itsability to oligomerize (5480ndash83) it was proposed thatdeaminase-independent inhibition could result from A3Gbinding to the ss template causing a lsquoroadblockrsquo toRT-catalyzed DNA elongation (637174)

To investigate whether A3A can also interfere with RTmovement along the template we used a modified versionof our reconstituted minus-strand transfer assay (6566)as described in lsquoMaterials and Methodsrsquo As shown inFigure 7 addition of 5 mM or even 10 mM A3A was notsufficient to reduce the levels of the extension product toany significant extent (compare with RT only reaction)independent of whether WT A3A or the E72Q deaminase-negative mutant was used This indicates that A3A isunable to directly block RT-catalyzed DNA extension

A3A has the potential to mutate DNA duringtranscription and SSB proteins could mitigate this effect

As discussed above A3A functions as a host restrictionfactor and plays a role in the host innate immuneresponse However it appears to also have an opposingbiological activity Thus its mutagenic potential may exerta detrimental effect on the cells where it is expressed es-pecially during cellular processes such as transcriptionwhich require transient opening of the duplex (strand sep-aration) To investigate this possibility we designed short

0

20

60

80

D

eam

inas

e P

rod

uct

40

100

A

0

20

60

80

L

uci

fera

se A

ctiv

ity

40

100

B

0 1 2 3 4 5

Empty

WTY132DD131YY132F

D131YY132D

Loop 7

Total Protein (microg)

120

140

WT Y132D D131Y Loop7

0

20

60

80

D

eam

inas

e P

rod

uct

40

100

D

0

20

60

80

L

uci

fera

se A

ctiv

ity

40

100

E

0 1 2 3 4 5

Empty

WTR69AI26G

R191D

F102A

Total Protein (microg)

120

140

WT R69A I26G R191D F102A

D131EY130F

P134A

Y132FD131YY132D

D131E Y130F P134A

C F

A3A A3A

Tubulin Tubulin

WT

Y132

DD

131Y

Y13

2F

D13

1YY

132D

Loop

7D13

1EY1

30F

WT

R69

AI2

6GR

191D

F102

A

P134

A

Figure 6 Deaminase and retrotransposition activities of A3A WT and non-active site mutants (A and D) Deaminase and (B and E) retrotran-sposition assays were performed as described in lsquoMaterials and Methodsrsquo (C and F) Western blot analysis of A3A WT and non-active site mutantexpression in 293T cells In the Loop 7 mutant A3A residues were changed to the corresponding residues in A3G Y132DD133QP134GL135R(Figure 1)

1104 Nucleic Acids Research 2014 Vol 42 No 2

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

(40 nt) dsDNA substrates containing unpaired regions atthe center to mimic transcription bubbles (TBs) of differ-ent sizes [1 nt (TB-1) 3 nt (TB-3) 5 nt (TB-5) and 9 nt (TB-9)] (Figure 8A) and tested their susceptibility to deamin-ation by WT A3A (Figure 8B)

As expected a dsDNA lacking any unpaired bases(Duplex) was essentially not deaminated (3) even atthe highest concentration of A3A used (100 nM) A par-tially dsDNA that contained a single unpaired dC nucleo-tide (TB-1) exhibited somewhat higher levels ofdeamination (16 and 23 with 50 and 100 nM A3Arespectively) Sequential addition of unpaired nucleotidessurrounding the central dC (TB-3 to TB-9) led toincreasing amounts of deaminase products up to 90with TB-9 at 100 nM A3A Moreover deaminationincreased as the concentration of A3A was raised from50 to 100 nM The results indicate that access of A3A tothe reactive dC in the transcription bubble becomes morefacile with increasing ss character and the more flexiblestructure of the larger bubbles These data clearlysupport the notion that A3A is capable of mutatinggenomic DNA during transcription in agreement withprevious studies by Love et al (14) using a cell-based tran-scription system

The data presented in Figure 8B raise an importantquestion is there a cellular mechanism that couldprevent possible A3A-induced damage to genomicDNA One possibility is that SSB proteins which coatssDNA regions of genomic DNA in the nucleus and areinvolved in maintenance of genome stability (84) blockA3A access to target sequences on unpaired DNAsReadily available SSB proteins including HIV-1 NC T4Gene 32 and E coli SSB were used to model this scenarioin vitro Deaminase assays were performed afterpreincubating the ssDNA substrate with different concen-trations of these SSB proteins Negative controls(no protein) and positive controls (A3A only) providedbasal and maximal activity for the final experimental con-ditions (Figure 8C) The maximal activity of A3A(positive control Lanes 2 6 and 10) remained essentiallythe same under different buffer conditions When ssDNAwas bound to HIV-1 NC no change in deamination was

observed whereas T4 Gene 32 or E coli SSB severelyreduced A3Arsquos ability to deaminate the ssDNA substrateThese results most likely reflect rapid onoff nucleic acidbinding kinetics of NC (7185) as opposed to the slowdissociation of the other two SSB proteins from ssDNA(86) which would block access of A3A to the DNAsubstrate

0Duplex TB-1

20

60

80

D

eam

inas

e P

rod

uct

40

100

TB-3 TB-5 TB-9

A

B

5prime3prime

ATTTCATTT

ATTTC ATTTTB-9

5prime3prime

TTCAT

TTC ATTB-5

5prime3prime

TCA

TC ATB-3

5prime3prime

C

CTB-1

01

20

60

80

D

eam

inas

e P

rod

uct

40

100

NC

120

T4 Gene 32 Ecoli SSB

2 3 4 5 6 7 8 9 10 11 12

C

Figure 8 Deamination of dC in ss regions of a 40-bp DNA duplex andthe effect of SSB proteins (A) Schematic representation of a series ofTBs in a ds nucleic acid Unpaired bases are located in the center of a40-bp DNA duplex that contains the TTCA sequence in the ss regionof one strand (B) Deaminase assay performed using duplexes (40 bp)containing TBs with different lengths of unpaired bases (1ndash9 nt) Theseduplexes were generated by heat annealing the ssDNA substrate(JL913) to oligonucleotides containing 1 nt (JL1088 TB-1) 3(JL1089 TB-3) 5 (JL1090 TB-5) and 9 (JL1091 TB-9) that are notcomplementary to the corresponding residues in the other DNA strand(C) Deaminase assay using the ssDNA substrate (JL913 180 nM) afterpreincubation with SSB proteins (HIV-1 NC T4 Gene 32 or E coliSSB each protein at 500 nM) for 15min at 37C before addition ofA3A and incubation for 1 h Bars 1 5 and 9 no proteins (negativecontrol) 2 6 and 10 A3A only (positive control) 3 7 11 and 4 8 12A3A and ssDNA preincubated with 25 or 5 mM SSB proteinrespectively

0

WT E72Q

20

60

80

E

xten

sio

n P

rod

uct

40

A3A (microM)RT +_ + + + +

_ _ 5 10 5 10

Figure 7 Effect of A3A on HIV-1 RT-catalyzed extension of a ()SSDNA oligonucleotide The bar graph shows the percent of DNAextension product without () or with (+) RT andor A3A Thepositive control reaction with RT only is shown as a white barReactions with RT contain either 5mM (black bar) or 10 mM (graybar) A3A (WT and E72Q mutant)

Nucleic Acids Research 2014 Vol 42 No 2 1105

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018

DISCUSSION

In the present work we report biochemical and mutagen-esis studies designed to elucidate the molecular propertiesof A3A and obtain mechanistic insights into A3A functionin light of the recently determined A3A NMR structure(16) Although it is well known that A3A deaminatesssDNA substrates it was of interest to evaluate from astructural perspective whether A3A could also use anssRNA substrate Within the human APOBEC familyA1 can deaminate RNA as required for tissue-specificediting of apolipoprotein B mRNA (87) and activation-induced deaminase (AID) a known ssDNA deaminasethat generates antibody diversity (9) has been implicatedin C to U editing of HBV nucleocapsid RNA (88) Incontrast A3G is not able to deaminate ssRNA (58)Here we directly demonstrate by NMR that A3A is

incapable of deaminating RNA (Figure 2A) in agreementwith an earlier observation (24) In addition it appearsfrom mapping the A3A-RNA binding interface(Figure 2C) that the cytosine ring in a 9-nt RNA doesnot permit optimal positioning for catalysis Some of theinteractions that were observed with a 9-nt ssDNA(having the same sequence as the 9-nt RNA) (16) areabsent suggesting a less intimate interaction betweenA3A and ssRNA (Figure 2C) compared with its inter-action with ssDNA (Figure 2D) While this article wasunder review Nabel et al published results showing thatdifferential puckering of the sugar moiety in ssDNA andssRNA substrates influences selective deamination ofssDNA by AID (89) Thus it seems possible that the in-ability of A3A to accommodate the cytosine in ssRNA inthe active site and the less intimate contact with ssRNAare related to the sugar pucker of the cytosine ribose ringWe have extended the previous analysis on the inter-

action with short ssDNA oligonucleotides to includessDNA substrates of varying lengths (up to 80 nt) andshow that longer oligonucleotides interact more avidlywith A3A (Figure 4A) This could be due to the greatercontribution of non-specific interactions as the length ofthe oligonucleotide is increased resulting in increasedbinding with the longer ssDNAs NMR analysisdemonstrated that although A3A binding to a 9-merssDNA is specific binding to a 15-mer ssDNA involvesboth specific and non-specific interactions (16)Comparison of the nucleic acid binding activities of

A3A with A3G in EMSA studies illustrates an interestingdifference between the properties of the two proteins Incontrast to one major band seen with A3A (Figure 4A)multiple product bands are observed in A3G gel-shiftassays likely due to formation of higher order complexeseven at an A3G concentration of 20 nM (58) These ob-servations may be related to the fact that A3Gmultimerizes especially in the presence of nucleic acids(5480ndash83) whereas A3A is monomeric in solution atleast at concentrations lt02mM (1416)The relationship between ssDNA length and deaminase

activity is also different for A3A and A3G At an A3Aconcentration of 100 nM deaminase activity is reduced asthe length of the ssDNA substrate is increased(Figure 4B) Non-specific interactions of A3A with these

longer ssDNAs at non-specific binding sites may contrib-ute to this inhibitory effect In addition the weak slidingand jumping activities of A3A on nucleic acid substrates(14) may interfere with its ability to locate the active sitemotif (TTCA) in the longer ssDNAs At a higher A3Aconcentration (eg 200 nM) however deaminase activityis independent of substrate length as A3A activity shouldbe saturated under conditions where the A3Asubstrateconcentration is 1 In contrast to A3A A3G exhibitsincreasing deaminase activity as the length of CCC-containing ssDNAs is increased from 15 nt to 60 nt (90)This result presumably reflects the fact that A3G has twoZn-binding domains one of which (the N-terminaldomain) exhibits strong nucleic acid binding activity(5891) whereas A3A is a single-domain protein

Structure-guided mutagenesis based not only on theA3A NMR structure but also on a model of thecomplex with the DNA target T2T1CA+1 (16) wasused to create alanine mutants of several A3A residuesthat interact with either T1 (P134) T2 (W104 andE138) or both (F102 and L135) These mutants exhibitreduced deaminase activity but the activity is not totallyabolished (Figures 3A and 6A) Thus individual aminoacid changes may not be sufficient for disrupting inter-actions that are important for substrate positioningduring catalysis However changing G105 whichcontacts T2 to alanine results in complete abrogationof catalytic activity (Figure 3A) either due to an unfavor-able conformational change in the binding site or loss ofthe overall structural integrity of the protein Interestinglydeletion of W104 and G105 results in only partial loss ofA3A catalytic activity (1733)

Studies of A3G-CD2 have shown that loop 7(313-RIYDDQGR-320) (Figure 1A) is an important de-terminant of deaminase specificity (preference for CCCAmotif) (6516970) In our A3AssDNA model (16)several residues (D131 Y132 D133 P134 and L135)that contact bases adjacent to the catalytic C reside inloop 7 (127-ARIYDYDPL-135) (Figure 1A) Althoughchanges of the last two amino acids in this loop (P134and L135) have only small effects on deaminase activity(Figures 6D and 3A respectively) substitutions ofresidues D131 and Y132 (Figure 6A) and D133 (13) leadto complete loss of catalytic function This indicates thatthe contributions of individual loop 7 residues to A3Adeaminase activity are not equivalent Interestinglyreplacing all A3G loop 7 residues with those found inA3A yields an active protein (92) whereas an A3A withan A3G loop 7 (Loop 7 mutant) exhibits complete loss ofenzyme activity (Figure 6A) This is most likely explainedby the fact that changing Y132 of A3A to the correspond-ing residue in A3G (D317) is detrimental to the deaminaseactivity of A3A (Y132D mutant Figure 6A) althoughA3G with Y317 is active (92)

Mutation of the Zn-binding residues (H70R C101S andC106S) and residues that directly contact the C in theTTCA motif (N57A E72A E72D and E72Q) abolishesA3A deaminase activity (Figure 5A) (101333) either bydestroying the structural integrity of the protein or byinterfering with the correct positioning of the cytosine inthe active site Interestingly changing the catalytic E72 to

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Q does not affect structural integrity (SupplementaryFigure S1) (16) or ability to bind ssDNA [Figure 4C(16)] only deaminase activity is affected Changes in theflexible loop 3 (F66A and G65A) only partially abrogatethe activity of A3A suggesting that the plasticity of thisloop is sufficient to allow binding andor catalysis

A key question that arises in studies of A3 proteins ishow deaminase activity is related to biological functionOne area of interest is the role of deamination in A3Arsquosinhibition of LINE-1 retrotransposition The data pre-sented here (Figures 5 and 6) indicate that A3A mutationsresult in three different phenotypes In some instancesLINE-1 restriction activity is present in the absence ofdeaminase activity For example E72D (Figure 5A andB) and D131E (Figure 6A and B) have no enzymaticactivity but exhibit close to WT levels of anti-LINE-1activity Other deaminase-negative mutants (eg Y130Fand Y132D [Figure 6A and B]) still restrict LINE-1 butto a lesser extent however increased expression of A3Aincreases restriction activity These findings are in accordwith earlier studies showing that no A3A-induced deamin-ation signatures could be detected in LINE-1 DNA(102627)

We have also characterized A3A mutants with the fol-lowing properties (i) presence of both deaminase and anti-LINE-1 activities (eg F102A and P134A [Figure 6D andE]) and (ii) absence of both activities (eg C101S andE72Q [Figure 5A and B]) Some examples of mutantswith phenotypes (i) and (ii) have also been reported byothers (101327) Importantly these results stronglysuggest that although the determinants of deaminase andanti-LINE-1 activities can overlap they are not identicalDeaminase activity is sensitive to even small changes in theactive site geometry and its immediate environmentHowever such changes may not significantly affect theanti-LINE-1 activity as in the case of the E72D andD131E mutants Thus taken together our data supportthe conclusion that deaminase and LINE-1 restrictionactivities are not linked We also considered the possibilitythat anti-LINE-1 activity might be related to the ability tobind ssDNA However the ssDNA binding experimentswith A3A mutant proteins show that despite their differ-ent anti-LINE-1 activities binding efficiency in each casewas equivalent to that of WT (Figure 4C) Thus underour conditions the data suggest that inhibition ofretrotransposition is not directly correlated with nucleicacid binding activity

In contrast to LINE-1 retrotransposition there isstrong evidence that the deamination activity of A3A isrequired for blocking replication of human retrovirusessuch as HIV-1 and HTLV-1 A3A deamination signatureswere observed in HIV-1 (1440) and HTLV-1 DNA (36)and it appears that DNA editing is the primary mode ofinhibition of these retroviruses As A3A binds only weaklyto nucleic acids (Figure 2 and 4) (16) and does notmultimerize (1416) it is not surprising that A3A fails toblock movement of RT during DNA synthesis (Figure 7)

In addition to deamination of viral ssDNA A3A hasalso been shown to edit cytosines present in genomic DNA(154445) and in foreign DNA plasmids transfected intomammalian cells (1215) How could an ssDNA

deaminase like A3A mutate ds genomic or foreignDNA Here we show that intact dsDNA clearly is not agood substrate for A3A (Figure 8B) (10) and that A3Aacts only on ss regions present in otherwise ds nucleic acid(Figure 8B) This suggests that genomic and foreign DNAin cells would only be accessible to A3A during transientstrand opening during processes such as transcription andDNA replication (93)APOBEC protein-mediated genomic DNA mutations

have been implicated in carcinogenesis (94ndash101) and arecent report has suggested a role for A3A in DNAbreak-associated mutations which are associated withbreast cancer (101) Therefore one may pose thequestion lsquohow is A3A regulated inside a cell so as notto cause undesirable damage to genomic DNArsquo Onescenario that is shown here is the possible involvementof SSB proteins with strong ss nucleic acid bindingaffinity and slow on-off rates that could block access togenomic DNA (Figure 8C) Interestingly there is a prece-dent for the mechanism we suggest in a recent report itwas shown that replication protein A a nuclear SSBprotein in yeast strongly inhibits A3G deaminaseactivity as well as enzyme processivity (102)Other mechanisms have also been proposed to explain

how genomic DNAmutations by A3Amight be preventedOne possibility involves reduction of A3A levels by TRIB 3(47) However this has been questioned in a recent study byLand et al (48) where TRIB 3 was reported to be ineffect-ive in preventing DNA damage by A3A Interestingly inthe same study localization of A3A within the cytoplasmof monocytic cells was shown to prevent genotoxicity sug-gesting that these cells have developed robust mechanismsto protect genomic DNA integrity However these mech-anisms might be compromised in pathological conditionssuch as cancer (103) and therefore need to be evaluatedindividually for different conditions and cellsIn summary the present study provides new insights

into the biological activities of human A3A by examin-ation of its deaminase and nucleic acid binding propertiesin the context of its 3D structure These properties definethe nature of A3Arsquos interaction with its nucleic acid sub-strates and are critical for its role as a host restrictionfactor and a genomic DNA mutator How cells regulateundesirable DNA editing by A3A and other human A3proteins is a major question for future investigation

NOTE ADDED IN PROOF

During the proof stage of this manuscript we becameaware of a paper by Pham et al which also reportedA3A deaminase activity on partially single-strandedDNA substrates (PhamP Landolph A Mendez CLi N and Goodman MF A biochemical analysislinking APOBEC3A to disparate HIV-1 restriction andskin cancer J Biol Chem doi 101074jbcM113504175)

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

Nucleic Acids Research 2014 Vol 42 No 2 1107

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ACKNOWLEDGEMENTS

The authors thank Tiyun Wu for valuable advice on theminus-strand transfer assay and for providing the RNAand DNA constructs Klara Post for helpful advice onEMSA gels Robert J Gorelick for purified NC proteinKlaus Strebel for antiserum used to detect A3A JasonConcel and Maria DeLucia for assistance in purificationof recombinant A3A Phil Greer and Doug Bevan forcomputer technical support and Michael J Delk forNMR instrumental support

FUNDING

Intramural Research Program at the National Institutes ofHealth [Eunice Kennedy Shriver National Institute ofChild Health and Human Development (to MM KHKH-R DS GN and JGL) and the National CancerInstitute HIV Drug Resistance Program and Center forCancer Research (to SH and GH)] and the NationalInstitutes of Health [Grant P50GM082251 to JA I-LBC-HB LMC and AMG] Funding for open accesscharge Intramural Program of NIHNICHD

Conflict of interest statement None declared

REFERENCES

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2 ChiuYL and GreeneWC (2008) The APOBEC3 cytidinedeaminases an innate defensive network opposing exogenousretroviruses and endogenous retroelements Annu Rev Immunol26 317ndash353

3 MalimMH (2009) APOBEC proteins and intrinsic resistance toHIV-1 infection Philos Trans R Soc Lond B Biol Sci 364675ndash687

4 DuggalNK and EmermanM (2012) Evolutionary conflictsbetween viruses and restriction factors shape immunity Nat RevImmunol 12 687ndash695

5 HolmesRK MalimMH and BishopKN (2007) APOBEC-mediated viral restriction not simply editing Trends BiochemSci 32 118ndash128

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10 ChenH LilleyCE YuQ LeeDV ChouJ NarvaizaILandauNR and WeitzmanMD (2006) APOBEC3A is a potentinhibitor of adeno-associated virus and retrotransposons CurrBiol 16 480ndash485

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30 LovsinN and PeterlinBM (2009) APOBEC3 proteins inhibitLINE-1 retrotransposition in the absence of ORF1p bindingAnn N Y Acad Sci 1178 268ndash275

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41 KoningFA GoujonC BaubyH and MalimMH (2011)Target cell-mediated editing of HIV-1 cDNA by APOBEC3proteins in human macrophages J Virol 85 13448ndash13452

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46 MussilB SuspeneR AynaudM-M GauvritA VartanianJ-Pand Wain-HobsonS (2013) Human APOBEC3A IsoformsTranslocate to the Nucleus and Induce DNA Double StrandBreaks Leading to Cell Stress and Death PloS One 8 e73641

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deaminase domain of the HIV-1 restriction factor APOBEC3GNature 452 116ndash119

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66 WuT DattaSAK MitraM GorelickRJ ReinA andLevinJG (2010) Fundamental differences between the nucleicacid chaperone activities of HIV-1 nucleocapsid protein and Gagor Gag-derived proteins biological implications Virology 405556ndash567

67 WuT Heilman-MillerSL and LevinJG (2007) Effectsof nucleic acid local structure and magnesium ions onminus-strand transfer mediated by the nucleic acid chaperoneactivity of HIV-1 nucleocapsid protein Nucleic Acids Res 353974ndash3987

68 YuQ KonigR PillaiS ChilesK KearneyM PalmerSRichmanD CoffinJM and LandauNR (2004) Single-strand specificity of APOBEC3G accounts for minus-stranddeamination of the HIV genome Nat Struct Mol Biol 11435ndash442

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70 KohliRM MaulRW GuminskiAF McClureRLGajulaKS SaribasakH McMahonMA SilicianoRFGearhartPJ and StiversJT (2010) Local sequence targeting inthe AIDAPOBEC family differentially impacts retroviralrestriction and antibody diversification J Biol Chem 28540956ndash40964

71 IwataniY ChanDSB WangF MaynardKS SugiuraWGronenbornAM RouzinaI WilliamsMC Musier-ForsythKand LevinJG (2007) Deaminase-independent inhibition of HIV-1reverse transcription by APOBEC3G Nucleic Acids Res 357096ndash7108

72 BishopKN VermaM KimE-Y WolinskySM andMalimMH (2008) APOBEC3G inhibits elongation of HIV-1reverse transcripts PLoS Pathog 4 e1000231

73 AdolphMB WebbJ and ChelicoL (2013) Retroviral restrictionfactor APOBEC3G delays the initiation of DNA synthesis byHIV-1 reverse transcriptase PloS One 8 e64196

74 ChaurasiyaKR McCauleyMJ WangW QualleyDF WuTKitamuraS GeertsemaH ChanDSB HertzA IwataniYet al (2013) Oligomerization transforms human APOBEC3Gfrom an efficient enzyme to a slowly dissociating nucleic acidbinding protein Nat Chem 6 28ndash33

75 BishopKN HolmesRK and MalimMH (2006) Antiviralpotency of APOBEC proteins does not correlate with cytidinedeamination J Virol 80 8450ndash8458

76 HolmesRK KoningFA BishopKN and MalimMH (2007)APOBEC3F can inhibit the accumulation of HIV-1 reversetranscription products in the absence of hypermutationComparisons with APOBEC3G J Biol Chem 282 2587ndash2595

77 LuoK WangT LiuB TianC XiaoZ KappesJ and YuXF(2007) Cytidine deaminases APOBEC3G and APOBEC3F interactwith human immunodeficiency virus type 1 integrase and inhibitproviral DNA formation J Virol 81 7238ndash7248

78 BelangerK SavoieM Rosales GerpeMC CoutureJ-F andLangloisMA (2013) Binding of RNA by APOBEC3G controlsdeamination-independent restriction of retroviruses Nucleic AcidsRes 41 7438ndash7452

79 GillickK PollpeterD PhaloraP KimE-Y WolinskySMand MalimMH (2013) Suppression of HIV-1 infection byAPOBEC3 proteins in primary human CD4+ T cells is associatedwith inhibition of processive reverse transcription as well asexcessive cytidine deamination J Virol 87 1508ndash1517

80 ChelicoL SachoEJ ErieDA and GoodmanMF (2008) Amodel for oligomeric regulation of APOBEC3G cytosinedeaminase-dependent restriction of HIV J Biol Chem 28313780ndash13791

81 HuthoffH AutoreF Gallois-MontbrunS FraternaliF andMalimMH (2009) RNA-dependent oligomerization ofAPOBEC3G is required for restriction of HIV-1 PLoS Pathog5 e1000330

82 McDougallWM OkanyC and SmithHC (2011) Deaminaseactivity on single-stranded DNA (ssDNA) occurs in vitro whenAPOBEC3G cytidine deaminase forms homotetramers and higher-order complexes J Biol Chem 286 30655ndash30661

83 ShlyakhtenkoLS LushnikovAY LiM LackeyL HarrisRSand LyubchenkoYL (2011) Atomic force microscopy studiesprovide direct evidence for dimerization of the HIV restrictionfactor APOBEC3G J Biol Chem 286 3387ndash3395

84 RichardDJ BoldersonE and KhannaKK (2009) Multiplehuman single-stranded DNA binding proteins function in genomemaintenance structural biochemical and functional analysis CritRev Biochem Mol Biol 44 98ndash116

85 CruceanuM GorelickRJ Musier-ForsythK RouzinaI andWilliamsMC (2006) Rapid kinetics of protein-nucleic acidinteraction is a major component of HIV-1 nucleocapsid proteinrsquosnucleic acid chaperone function J Mol Biol 363 867ndash877

86 PantK KarpelRL RouzinaI and WilliamsMC (2004)Mechanical measurement of single-molecule binding rateskinetics of DNA helix-destabilization by T4 gene 32 protein JMol Biol 336 851ndash870

87 TengB BurantCF and DavidsonNO (1993) Molecularcloning of an apolipoprotein B messenger RNA editing proteinScience 260 1816ndash1819

88 LiangG KitamuraK WangZ LiuG ChowdhuryS FuWKouraM WakaeK HonjoT and MuramatsuM (2013)RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase Proc Natl Acad Sci USA 1102246ndash2251

89 NabelCS LeeJW WangLC and KohliRM (2013) Nucleicacid determinants for selective deamination of DNA over RNAby activation-induced deaminase Proc Natl Acad Sci USA110 14225ndash14230

90 ChelicoL PhamP CalabreseP and GoodmanMF (2006)APOBEC3G DNA deaminase acts processively 30 ndashgt 50 onsingle-stranded DNA Nat Struct Mol Biol 13 392ndash399

91 NavarroF BollmanB ChenH KonigR YuQ ChilesKand LandauNR (2005) Complementary function of the twocatalytic domains of APOBEC3G Virology 333 374ndash386

92 RathoreA CarpenterMA DemirO IkedaT LiMShabanNM LawEK AnokhinD BrownWL AmaroREet al (2013) The local dinucleotide preference of APOBEC3Gcan be altered from 50-CC to 50-TC by a single amino acidsubstitution J Mol Biol 425 4442ndash4454

93 ChahwanR WontakalSN and RoaS (2010) Crosstalkbetween genetic and epigenetic information through cytosinedeamination Trends Genet 26 443ndash448

94 Nik-ZainalS AlexandrovLB WedgeDC Van LooPGreenmanCD RaineK JonesD HintonJ MarshallJStebbingsLA et al (2012) Mutational processes molding thegenomes of 21 breast cancers Cell 149 979ndash993

95 RobertsSA SterlingJ ThompsonC HarrisS MavDShahR KlimczakLJ KryukovGV MalcEMieczkowskiPA et al (2012) Clustered mutations in yeast andin human cancers can arise from damaged long single-strandDNA regions Mol Cell 46 424ndash435

96 AlexandrovLB Nik-ZainalS WedgeDC AparicioSAJRBehjatiS BiankinAV BignellGR BolliN BorgABoslashrresen-DaleA-L et al (2013) Signatures of mutationalprocesses in human cancer Nature 500 415ndash421

97 BurnsMB LackeyL CarpenterMA RathoreA LandAMLeonardB RefslandEW KotandeniyaD TretyakovaNNikasJB et al (2013) APOBEC3B is an enzymatic source ofmutation in breast cancer Nature 494 366ndash370

98 BurnsMB TemizNA and HarrisRS (2013) Evidence forAPOBEC3B mutagenesis in multiple human cancers NatGenet 45 977ndash983

99 NowarskiR and KotlerM (2013) APOBEC3 cytidinedeaminases in double-strand DNA break repair and cancerpromotion Cancer Res 73 3494ndash3498

100 RobertsSA LawrenceMS KlimczakLJ GrimmSAFargoD StojanovP KiezunA KryukovGV CarterSLSaksenaG et al (2013) An APOBEC cytidine deaminasemutagenesis pattern is widespread in human cancers NatGenet 45 970ndash976

101 TaylorBJ Nik-ZainalS WuYL StebbingsLA RaineKCampbellPJ RadaC StrattonMR and NeubergerMS(2013) DNA deaminases induce break-associated mutationshowers with implication of APOBEC3B and 3A in breastcancer kataegis eLife 2 e00534

102 LadaAG WaisertreigerIS-R GrabowCE PrakashABorgstahlGEO RogozinIB and PavlovYI (2011)Replication protein A (RPA) hampers the processive action ofAPOBEC3G cytosine deaminase on single-stranded DNA PloSOne 6 e24848

103 HungM-C and LinkW (2011) Protein localization in diseaseand therapy J Cell Sci 124 3381ndash3392

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69 CarpenterMA RajagurubandaraE WijesingheP andBhagwatAS (2010) Determinants of sequence-specificity withinhuman AID and APOBEC3G DNA Repair 9 579ndash587

70 KohliRM MaulRW GuminskiAF McClureRLGajulaKS SaribasakH McMahonMA SilicianoRFGearhartPJ and StiversJT (2010) Local sequence targeting inthe AIDAPOBEC family differentially impacts retroviralrestriction and antibody diversification J Biol Chem 28540956ndash40964

71 IwataniY ChanDSB WangF MaynardKS SugiuraWGronenbornAM RouzinaI WilliamsMC Musier-ForsythKand LevinJG (2007) Deaminase-independent inhibition of HIV-1reverse transcription by APOBEC3G Nucleic Acids Res 357096ndash7108

72 BishopKN VermaM KimE-Y WolinskySM andMalimMH (2008) APOBEC3G inhibits elongation of HIV-1reverse transcripts PLoS Pathog 4 e1000231

73 AdolphMB WebbJ and ChelicoL (2013) Retroviral restrictionfactor APOBEC3G delays the initiation of DNA synthesis byHIV-1 reverse transcriptase PloS One 8 e64196

74 ChaurasiyaKR McCauleyMJ WangW QualleyDF WuTKitamuraS GeertsemaH ChanDSB HertzA IwataniYet al (2013) Oligomerization transforms human APOBEC3Gfrom an efficient enzyme to a slowly dissociating nucleic acidbinding protein Nat Chem 6 28ndash33

75 BishopKN HolmesRK and MalimMH (2006) Antiviralpotency of APOBEC proteins does not correlate with cytidinedeamination J Virol 80 8450ndash8458

76 HolmesRK KoningFA BishopKN and MalimMH (2007)APOBEC3F can inhibit the accumulation of HIV-1 reversetranscription products in the absence of hypermutationComparisons with APOBEC3G J Biol Chem 282 2587ndash2595

77 LuoK WangT LiuB TianC XiaoZ KappesJ and YuXF(2007) Cytidine deaminases APOBEC3G and APOBEC3F interactwith human immunodeficiency virus type 1 integrase and inhibitproviral DNA formation J Virol 81 7238ndash7248

78 BelangerK SavoieM Rosales GerpeMC CoutureJ-F andLangloisMA (2013) Binding of RNA by APOBEC3G controlsdeamination-independent restriction of retroviruses Nucleic AcidsRes 41 7438ndash7452

79 GillickK PollpeterD PhaloraP KimE-Y WolinskySMand MalimMH (2013) Suppression of HIV-1 infection byAPOBEC3 proteins in primary human CD4+ T cells is associatedwith inhibition of processive reverse transcription as well asexcessive cytidine deamination J Virol 87 1508ndash1517

80 ChelicoL SachoEJ ErieDA and GoodmanMF (2008) Amodel for oligomeric regulation of APOBEC3G cytosinedeaminase-dependent restriction of HIV J Biol Chem 28313780ndash13791

81 HuthoffH AutoreF Gallois-MontbrunS FraternaliF andMalimMH (2009) RNA-dependent oligomerization ofAPOBEC3G is required for restriction of HIV-1 PLoS Pathog5 e1000330

82 McDougallWM OkanyC and SmithHC (2011) Deaminaseactivity on single-stranded DNA (ssDNA) occurs in vitro whenAPOBEC3G cytidine deaminase forms homotetramers and higher-order complexes J Biol Chem 286 30655ndash30661

83 ShlyakhtenkoLS LushnikovAY LiM LackeyL HarrisRSand LyubchenkoYL (2011) Atomic force microscopy studiesprovide direct evidence for dimerization of the HIV restrictionfactor APOBEC3G J Biol Chem 286 3387ndash3395

84 RichardDJ BoldersonE and KhannaKK (2009) Multiplehuman single-stranded DNA binding proteins function in genomemaintenance structural biochemical and functional analysis CritRev Biochem Mol Biol 44 98ndash116

85 CruceanuM GorelickRJ Musier-ForsythK RouzinaI andWilliamsMC (2006) Rapid kinetics of protein-nucleic acidinteraction is a major component of HIV-1 nucleocapsid proteinrsquosnucleic acid chaperone function J Mol Biol 363 867ndash877

86 PantK KarpelRL RouzinaI and WilliamsMC (2004)Mechanical measurement of single-molecule binding rateskinetics of DNA helix-destabilization by T4 gene 32 protein JMol Biol 336 851ndash870

87 TengB BurantCF and DavidsonNO (1993) Molecularcloning of an apolipoprotein B messenger RNA editing proteinScience 260 1816ndash1819

88 LiangG KitamuraK WangZ LiuG ChowdhuryS FuWKouraM WakaeK HonjoT and MuramatsuM (2013)RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase Proc Natl Acad Sci USA 1102246ndash2251

89 NabelCS LeeJW WangLC and KohliRM (2013) Nucleicacid determinants for selective deamination of DNA over RNAby activation-induced deaminase Proc Natl Acad Sci USA110 14225ndash14230

90 ChelicoL PhamP CalabreseP and GoodmanMF (2006)APOBEC3G DNA deaminase acts processively 30 ndashgt 50 onsingle-stranded DNA Nat Struct Mol Biol 13 392ndash399

91 NavarroF BollmanB ChenH KonigR YuQ ChilesKand LandauNR (2005) Complementary function of the twocatalytic domains of APOBEC3G Virology 333 374ndash386

92 RathoreA CarpenterMA DemirO IkedaT LiMShabanNM LawEK AnokhinD BrownWL AmaroREet al (2013) The local dinucleotide preference of APOBEC3Gcan be altered from 50-CC to 50-TC by a single amino acidsubstitution J Mol Biol 425 4442ndash4454

93 ChahwanR WontakalSN and RoaS (2010) Crosstalkbetween genetic and epigenetic information through cytosinedeamination Trends Genet 26 443ndash448

94 Nik-ZainalS AlexandrovLB WedgeDC Van LooPGreenmanCD RaineK JonesD HintonJ MarshallJStebbingsLA et al (2012) Mutational processes molding thegenomes of 21 breast cancers Cell 149 979ndash993

95 RobertsSA SterlingJ ThompsonC HarrisS MavDShahR KlimczakLJ KryukovGV MalcEMieczkowskiPA et al (2012) Clustered mutations in yeast andin human cancers can arise from damaged long single-strandDNA regions Mol Cell 46 424ndash435

96 AlexandrovLB Nik-ZainalS WedgeDC AparicioSAJRBehjatiS BiankinAV BignellGR BolliN BorgABoslashrresen-DaleA-L et al (2013) Signatures of mutationalprocesses in human cancer Nature 500 415ndash421

97 BurnsMB LackeyL CarpenterMA RathoreA LandAMLeonardB RefslandEW KotandeniyaD TretyakovaNNikasJB et al (2013) APOBEC3B is an enzymatic source ofmutation in breast cancer Nature 494 366ndash370

98 BurnsMB TemizNA and HarrisRS (2013) Evidence forAPOBEC3B mutagenesis in multiple human cancers NatGenet 45 977ndash983

99 NowarskiR and KotlerM (2013) APOBEC3 cytidinedeaminases in double-strand DNA break repair and cancerpromotion Cancer Res 73 3494ndash3498

100 RobertsSA LawrenceMS KlimczakLJ GrimmSAFargoD StojanovP KiezunA KryukovGV CarterSLSaksenaG et al (2013) An APOBEC cytidine deaminasemutagenesis pattern is widespread in human cancers NatGenet 45 970ndash976

101 TaylorBJ Nik-ZainalS WuYL StebbingsLA RaineKCampbellPJ RadaC StrattonMR and NeubergerMS(2013) DNA deaminases induce break-associated mutationshowers with implication of APOBEC3B and 3A in breastcancer kataegis eLife 2 e00534

102 LadaAG WaisertreigerIS-R GrabowCE PrakashABorgstahlGEO RogozinIB and PavlovYI (2011)Replication protein A (RPA) hampers the processive action ofAPOBEC3G cytosine deaminase on single-stranded DNA PloSOne 6 e24848

103 HungM-C and LinkW (2011) Protein localization in diseaseand therapy J Cell Sci 124 3381ndash3392

1110 Nucleic Acids Research 2014 Vol 42 No 2

Downloaded from httpsacademicoupcomnararticle-abstract42210951028425by gueston 13 February 2018