CHAPTER3

CLONING, PURIFICATION &

CHARACTERIZATION OF LIGASE A & DOMAINS

Cha ter 3

3.1 Introduction

Macromolecular crystallography offers an important tool to study the mechanistic

and functional details of proteins at a molecular level. Obtaining large amounts of protein

is a typical requirement of any crystallographic study in order to analyze the different

permutations and combinations of crystallization parameters. Current advances in

recombinant DNA technology have enabled us to explore different expression systems to

optimize conditions for production of good quantity of required proteins for structural I

biophysical studies.

In this chapter, we describe the results of cloning, overexpression and purification,

and characterization of full length Rv3014c as well as its mutants.

3.2 Results and Discussion

3.2.1 Sequence alignment studies

Rv3014c has been annotated as a putative NAD+ -dependent DNA ligase

(LigA) in the Mycobacterium tuberculosis genome databases such as Tuberculist

(http://genolist.pasteur.fr/Tuberculist/) (Cole et a!., 1998). BLASTP searches against

the protein sequence of Rv3014c resulted in several hits with 13 hits having 0.0 e

values, all relating it to NAD+ ligases. The MtuLigA protein (691 amino acids) has

extensive sequence homology (over 40 % sequence identity) and possesses the motifs

and domains typical to that of other well characterized NAD+ -dependent ligases from

organisms such as Bacillus subtilis, Escherichia coli and Haemophilus injluenzae.

The N-terminal region possesses five motifs (I, III, Ilia, IV and V) (Fig. 3.1) that

define the polynucleotide ligase/mRNA capping enzyme superfamily of covalent

nucleotidyltransferases (Shuman and Lima, 2004). Motif I (KXDG) contains the

lysine nucleophile to which AMP becomes covalently linked in the first step of

ligation reaction (Odell et a!., 2000; Tomkinson et a!., 1991 ). Motifs III, lila, IV, and

V contain conserved side chains that contact AMP and are essential for the

nucleotidyl transfer reactions (Subramanya et a!., 1996; Odell et a!., 2000, Shuman

and Ru, 1995). The C-terminal segment has sequence homology to the oligomer

binding (OB) domain, a tetra-cysteine domain that binds with Zn atom, helix-hairpin­

helix domain (HhH motif) and BRCAI like C-terminus domain (BRCT domain)

which is a signature sequence of C-terminal region of all eubacterial NAD+ ligases.

The OB-fold domain includes nucleotidyltransferase motif VI, which contacts the ~

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and y phosphates of the NTP substrate (Hakansson et al., 1997) and which IS

uniquely required for step 1 of the ligase reaction (Sriskanda and Shuman, 1998).

Secondary structure analysis also pointed to the homology between

Mycobacterium tuberculosis LigA sequence with other known ligases from Escherichia

coli, Salmonella typhimurium, Bacillus subtilis, Bacillus stearothermophilus and Thermus

filiformis (Konrad et al., 1973; Gottesman eta!., 1973; Park eta!., 1989; Petit and Ehrlich,

2000; Singleton et al., 1999; Timson and Wigley et al., 1999; Lee et al., 2000).

Till date, crystal structures of full length Thermus filiformis NAD+ ligase (Lee et

al., 2000), the nucleotidyltransferase domains of Bacillus stearothermophilus (Singleton et

a!., 1999) and Enterococcusfaecalis (Gajiwala and Pinko, 2004) are known. The sequence

of Rv3014c was aligned to the protein sequence of the structurally defined Thermus

filiformis NAD+ -dependent ligase to identify the position of different domains (Fig. 3.1).

Based on the sequence alignment (Fig. 3.1 and Fig. 3.2), we could demarcate the four

distinct domains of MtuLigA viz. adenylation or nucleotidyltransferase domain (1-328),

oligomer binding domain (329 -414), Zn finger and helix-hairpin-helix domain (415 -

605) and BRCT domain (BRCA1 like C-terminus, 606- 691).

In order to probe the functional roles of the domains, oligos for three C­

terminal deletion mutants, based on the above analysis, were constructed (Section 2.2,

chapter 2). Organization and nomenclature of full length and deletion ·mutants are as

follows.

MtuLigA Full length DNA ligase.

MtuLigAc Full length with C-terminal Histidine tag.

MtuLigAl Residues 1 - 605, BRCT domain is excluded.

MtuLigA2 Residues 1 - 439, BRCT and helix-hairpin-helix domain excluded.

MtuLigA3 Residues 1 - 328, only nucleotidyltransferase domain retained.

114

lOGS E . coli Mt b

lOGS E . coli Mtb

lOGS E . coli Mtb

lOGS E . coli Mtb

lOGS E. coli Mtb

lOGS E . coli Mtb

lOGS E . coli Mtb

lOG S E . col i Mtb

lOGS E . coli Mtb

lOGS E. coli Mtb

lOGS E . coli Mtb

lOGS E. coli Mtb

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-------MTREEARRRINELROLIRYHNYRYYVLAOPEISOAEYORLLRELKELEERFPE -- - ------MESIEQQLTELRTTLRHHEYLYHVMOAPEIPOAEYORLMRELRELETKHPE VSS POAOQTAPEVLRQWQALAEEVREHQFRYYVROAPII SOAEFOELLRRLEALEEQHPE

: * * : : * : * * * *** · * * •* * ** . ** . . . ... Adenylation Domain

FKSPOSPTEQVGARPLEPTFRPVRHPTRMYSLONAFTYEEVLAFEERLER-- EAEAPSLY LITPOSPTQRVGAAPLA- AFSQIRHEVPMLSLONVFOEESFLAFNKRVQORLKNNEKVTW LRTPOSPTQLVGGAGFATOFEPVOHLERMLSLONAFTAOELAAWAGRIHA-- EVGOAAHY .. *****. ** . * * **** * * . . . .

TVEHKVOGLSV - LYYEEGVWSTGS--GOGEVGEEVTQNLLTIPTIPRRLKG--- - - VPOR CCELKLOGLAVSILYENGVLVSAATRGOGTTGEOITSNVRTIRAIPLKLHGEN--- IPAR LCELKIOGVALSLVYREGRLTRASTRGOGRTGEOVTLNARTIAOVP ERLTPGOOYPVPEV

* * · ** · ... * · * . . . . . . . *** ** : : * * HI lil a

LEVRGEVYMPIEAFLRLNEELEERGEKVFKNPRNAAAGSLRQKOPRVTAKRGLRATFYAL LEVRGEVFLPQAGFEKINEOARRTGGKVFANPRNAAAGSLRQLOPRITAKR-- PLTFFCY LEVRGEVFFRLOOFQALNASLVEEGKAPFANPRNSAAGSLRQKOPAVTARR-- RLRMI CH ******* :: * **** · ******* ** · ** · * . . . . .

GLGLGLEESGLKSQYELLLWLKEKGFPVEHCYEKALGAEGVEEVYRRGLAQRHALPFEAO GVGVLEGGELPOTHLGRLLQFKKWGLPVSORVTLCESAEEVLAFYHKVEEORPTLGFOIO GLGHVEG- FRPATLHQAYLALRAWGLPVSEHTTLATOLAGVRERIOYWGEHRHEVOHEIQ * · * * .. * · ** ... *

IV v 3 17 GVVLKLOOLTLWGELGYTARAPRFALAYKFP GVVIKVNSLAQQEQLGFVARAPRWAVAFKFPAQEQMTFVROVEFQVGRTGAITPVARLEP GVVVKVOEVALQRRLGSTSRAPRWAIAYKYPPEEAQTKLLOIRVNVGRTGRITPFAFMTP * * * : * : : . : : ** . : **** : * : * : * : * . : * ·** *** · **

RWP VHVAGVL VS NATLHNAOEIERLGLRIGOKVVIRRAGOVIPQVV NVV LSERPEOTREVV FP VKVAGSTVGQATLHNAS EIKRKGVL I GOTVVIRKAGOVIPEVLGPVVELROGS EREFIMP

* : . . : *** * :::: ** . *** : * :

Zn fin ger (VI) -+30 Helix-EACPECG --HRLV!'. EG- KVHRCPN-PLC PAKRFEAIRHYASRKAMOIEGLGEKLIERLLE THCPVCGSOVERVEGE- AVARCTGGLICGAQRKESLKHFVSRRAMOVOGMGOKIIOQLVE TT~PE~GSPLAPEKEGOAOIR~PNARG~PGQLRERVFHVASRNGLOIEVLGYEAGVALLQ

** ** * . . * . * .. : * :: : * :

Hairpin- Helix Domain KGLVROVAOLYHLRKEOLLGLERMGEKS------AQNLLRQIEESKHRGLERLLYALGLP KEYVHTPAOLFKLTAGKLTGLERMGPKS------AQNVVNALEKAKETTFARFLYALGIR AKVIAOEGELFALTEROLLRTOLFRTKAGELSANGKRLLVNLOKAKAAPLWRVLVALSIR

: * . * * * . :

GVGEVLARNLARRFGTMORLLEASLEELIEVEEVGELTARAILETLKDPAFRDLVRRLKE EVGEATAAGLAAYFGTLEALEAASI EELQKVPOVG I VVASHVHNFFAEESNRNVI SELLA HVGPTAARALATEFGSLOAIAAASTOQLAAVEGVGPTIAAAVTEWFAVOWHREI VOKW RA

** * : : : . 58 1 BRCA I like C-Terminus (BRCT Domain)

AGVSM ES -- - - - l:EEVS OLLSGLT FVLTGELS PP - REEVKALLGRLGAFVT OSVS Rl:TS Y EGVHWPAPI VINAEEIOSPFAGKTVV LTGSLSQMSROOAKARLVELGAKVAGSVSKKTDL AGVRMVDE--- RDESVPRTLAGLTIVVTGSLTGFSROOAKEAIVARGGKAAGSVSKKTNY

* .: ··* * * · ** * · . . . . . .

LVVGENPGSfLEFARALGVAVLTEEEFWRFLFEKGAPVP.~. 6 6 7

VIAGEAAGSKLAKAQELGIEVIOEAEMLRLLGS------- 671 VVAGDSPGSKYOKAVELGV PILOEOG FRRLLAOGPASRT - 691 .. * . * . *

* . * .:. *** : **

Fig 3.1: Sequence alignment of MtuLigA with lOGS (PDB ID ofT filiform is ligaseA) and well -characterized LigA from E. coli . Six conserved sequence e lements (underlined) present among members of the covalent nuc leotidy ltransferase superfamily including A TP -dependent ligases, RNA and tRNA and the eukaryotic mRNA guany ly l transferases are represented by I, ILl , lila, 1V, V, and VI. Different domains (written above the sequences) are represented by different co lors.

65

Tfili MtuLigA3 Est Efa

T fi l i MtuLigA3 Est Efa

Cha fer 3

-------MTREEARRRINELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPE 53 MGSPDADQTAPEVLRQWQALAEEVREHQFRYYVRDAPIISDAEFDELLRRLEALEEQHPE 60 --MDRQ-----QAERRAAELRELLNRYGYEYYVLDRPSVPDAEYDRLMQELIAIEEQYPE 53 --MEQQPLTLTAATTRAQELRKQLNQYS HEYYVKDQPSV EDYVYDRLYKELVDIETEFP D 58

*** . * ......

FKSPDSPTEQVGARPLEPTFRPVRHPTRMYSLDNAFTYEEVLAFEEP.L EPJI.LGP!<RPFL Y 11 3 LRTPDSPTQLVGGAGFATDFEPVDHLERMLSLDNAFTADELAAWAGRIHAE VG --DAAHY 118 LKTSDSPTQRIGGPPLEA- FRKVA HRVPMMSLANAFGEGDLRDFDRRVRQEVG-- - EAAY 109 LITPDSPTQRVGGKVLSG- FE KA PHDIPMYS LNDGFSKEDIFAFDERVRKAIGK- -PVAY 115 : :. ****: : *. * * * * : . * * . . *

I Tfili TVEH r· LSV!JLYYEEGVLVFGATRGDGEVGEEVTQNLLTI PTI PPP.LF:G-----VPDR 168 MtuLigA3 LCEL'·:· VALSLVYREGRLTRASTRGDGRTGEDVTLNARTIADVPERLTPGDDYPVPEV 178 Est VCEL ~. - " LAVSVRYEDGYFVQGATRGDGTTGEDITENLKTIRSLPLRLKE- - ----PVS 163 Efa CC EL' '-[ LAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMRLTE------PI S 169

· ** ··· . * · * . · ***** ** ·· * * * · · * . . ....

III Ilia Tfili MPIEA FLRLNEELEERGEKVFENPRIJAAAGSLR KDPRVTAf'RG-- -;:- 228 MtuLigA3 r ' P,E FRLDDFQALNASLVE EGKAPFANPRNSAAGSLRQKDPAVTARRR.,tl. t1'- 238 Bst , ,-~-MPKAS FLRLNEERKARGEELFANPRNAAAGSLRQLDPKVAASRQ "-F· 223 Efa ""r MPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRN' ,-fLI~· 229

Tfili Mtu Li gA3 Bst Efa

T fili MtuLigA3 Bst Efa

** .* : * **** · ******* * . *

GLEESGLKSQYELLLWLKEKGFPVEHGYEKALGAEGVEEVYRRFLAQRHALPFEA" - VEGFRPATLHQAYLALRAWGLPVS EHTTLATDLAGVRERID YWG EHRHEVDHEI '

.. 1 - AEALGIASHSEALDYLQALGFKVNPERRRCANIDEVIAFVSEWHDKRPQLPYEI '[ -FGPMKAKTQFEALEELSAIG FRTN PERQLCQSIDEVWAYIEEYHEKRSTLPYEI

* * : ..

v LDDLALWRELGYTJl.RAPP.F . FPAEEKETRLL 326

I V DEVALQRRLGST SRAPRW o=~- YPPE------- 328 VDSFAQQRALGATAK SPRW .::."'' 'FPAEEVVTT-- 318

" VN EFALQDELGFTVKAPRW FPPEEAETVLE 326 * : * : : .. :: ** : * : *** : * . *

Fig 3.2: Nucleotidyltransferase domain of bacterial LigaseA

IV 288 297 282 288

The amino acid sequence of the nucleotidyltransferase domain of M tuberculosis LigaseA (MtuLigA3) is aligned to the sequences of Li-gA from, B. stearothermophilus (Bst), T filiformis (Tfi), and E. faecalis (Efa). It further consists of two sub-domains Ia and Ib shown in blue and red in TfiLigA sequence. Five of the six conserved nucleotidyl transferase motifs I, III, Ilia, IV, and V lie in this domain (depicted in light blue). Gaps in the alignment are indicated by dashes. Conserved residues are indicated by *. Sequences were retrieved from www.rcsb.org. PDB IDs of Thermus fil~formis, Bacillus strearothermophilus and Enterococcus faecal is LigA are 1 V9P (earlier I DGS), 1 B04 and 1 TAE respectively.

66

3.2.2 Cloning, overexpression, purification and activity

3.2.2.1 Clonihg of full-length ligases and deletion mutants

Cha ter 3

The gene of Rv3014c of M. tuberculosis H37Rv is 2076 bp long and was amplified

by PCR using two different sets of oligos for native and C-terminal his-tagged protein.

These were cloned into T7 RNA polymerase-based bacterial expression vectors, pET21 d

and pET4la (Section 2.3.1, Chapter 2) (Fig. 3.3 A). The resultant constructs were

designated MtuLigA and MtuLigAc.

Similarly, deletion mutants were PCR amplified and cloned into TS RNA

polymerase-based bacterial expression vector pQ£60 between Nco! and BamHI with their

respective set of oligos which introduced CL186, C6252 and C6363 deletions respectively

for MtuLigA1, MtuLigA2 and MtuLigA3 (Section 2.3.2, Chapter 2). All the deletion

mutants contained 6x histidine tag at C-terminal. Clones were confirmed through

restriction digestion (Fig. 3.3 B). Incorporation of Nco! in forward oligo in MtuLigAs and

in deletion mutants leads to replacement of first two amino acids of sequence from valine

and serine to methionine and glycine while incorporation of Nde! in MtuLigAc leads to

replacement of valine to methionine. Integrity of all the inserts were verified by

sequencing and none of them contained any mutation.

3.2.2.2 Overexpression and purification

The expression plasmids were introduced into E.coli BL21 (DE3), a strain that

contains T7 RNA polymerase gene under the control of lacUVS promoter. While MtuLigA

and MtuLigAl were optimized for growth and induction at 37°C for 3 h (Section 2.3.1 ,

Chapter 2); MtuLigAc, MtuLigA2 and MtuLigA3 were grown and induced at 30°C (Section

2.3.2, Chapter 2) as these proteins were insoluble at 37°C. All the proteins were optimally

soluble (60 - 70%) and overexpressed under these conditions and a prominent - 75 kDa

polypeptide was detected in case of MtuLigA and MtuLigAc and - 67 kDa, 48kDa and 36

kDa in case of MtuLigA 1, A2 and A3 respectively by SDS-P AGE in whole cell extracts of

IPTG induced bacteria. These polypeptides were not present in uninduced samples or when

bacteria containing the pET or pQE vectors (as the case may be) alone were induced with

IPTG (Fig. 3.4 A, B and C). MtuLigA was purified using a strong anion exchanger (Q

sepharose) followed by affinity separations using HiTrap heparin-sepharose and blue­

sepharose columns, which are specific for DNA and NAD+ binding proteins. SDS-PAGE

analysis of the blue-sepharose eluate fraction showed that the preparation was essentially

homogenous with respect to the- 75kDa MtuLigA polypeptide (Fig. 3.4 A).

67

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1 2 3 4 5

10 8 6 5 4

3

2

1.5

0 .5

(A)

1 2 3 4 5 6 7 10

8 (l

5 -1

3

2

1. 5

(B)

Fig 3.3: Cloning of M. tuberculosis ligase A and its deletion mutants

(A) Cloning of ligaseA as native and his-tagged protein, Lane 1, PCR product of MtuLigA; Lane 2, Restriction digestion of ligA clone in pET2 1 d; Lane 3, PCR product of MtuLigAc; Lane 4, Restriction digestion of ligA clone in pET -11 a; Lane 5, 1 kilobase DNA ladder (Amersham).

(B) Cloning of C-terminal deletion mutants of ligaseA, Lane 1, 3 & 5, PCR products of MtuLigA 1, MtuLigA2 & MtuLigA3 ; Lane 2, 4 & 6, Restriction digestion of the same in that order; Lane 7, 1 kilobase DNA ladder (Amersham).

68

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The his-tagged MtuLigAc (Fig. 3.4 B) and deletion mutants viz. MtuLigA I,

MtuLigA2 and MtuLigA3 were affinity purified using Ni - chelating chromatography to

obtain high level of purity as observed through SDS-PAGE analysis (Fig. 3.4 C). The his­

tag facilitated rapid purification of the recombinants by absorption to an immobilized

nickel resin and subsequent elution with a buffer containing imidazole.

After this step proteins were precipitated using ammonium sulphate saturation.

Cut-off values of saturation for full length were optimized up to 65 %, for MtuLigA 1 60 %

while for other two mutants saturation reaches at 50 %. After this step, all the proteins

were dissolved in minimum volume of buffer C (50 mM Tris-HCl, pH 8.0, 50 mM NaCl,

2 mM mercaptoethanol, 1 mM EDTA) and further purified using gel filtration

chromatography.

3.2.2.3 Gel filtration analysis

Gel filtration chromatography was used to ascertain the oligomeric state of M.

tuberculosis LigaseA under standard experimental conditions. Both MtuLigA and

MtuLigAc show monomeric behaviour. (Gong et al., 2004). We used pre-packed

Superdex 200 high-resolution column in all the purifications to remove the trace

contamination ensuring high level of purification. Every protein shows a

characteristic gel permeation profile mainly depending on its molecular wt., strokes

radius etc. The column used to ascertain the mol. wt. was calibrated with known

molecular weight marker proteins and calibration graph of Ye/ V0 was plotted against

the logarithm of mol. wt. of the corresponding proteins. Ye and V 0 represent elution

volume of the protein and void volume of the column respectively. Blue dextran

(2000 kDa) was used to determine the void volume. MtuLigA and MtuLigAc eluted

at 13.90 ml and 13.70 ml respectively (Fig. 3.5 A) corresponding to 77.96 kDa and

80.61 kDa, which is in good agreement with theoretical values 75.26 kDa and 79 .37

kDa respectively.

MtuLigA1, MtuLigA2 and MtuLigA3 eluted at 14.15 ml, 14.20 ml and 15.10 ml,

all of them in a good agreement with the calibration curve (Fig. 3.5 B).

3.2.2.4 Ligase and adenylyltransferase activity

DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at nicks

in duplex DNA via 3 sequential nucleotidyl transfer reaction (Lehman, 1974; Engler and

Richardson, 1982). In step one, attack on the a-phosphorous of ATP or NAD+ by ligase

results in release of pyrophosphate or nicotinamide mononucleotide and formation of a

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UI QS HS BS GF Ul L WI W2 p Mr

116

66.

45

35

25

18. 14.

MtuLigA MtuLigAc (A) (B)

UI I L WI W2 P Ul I L WI W2 P Ul I L WI W2 p Mr

116

66

45 35

25 18

MtuLigAl MtuLigA2 MtuLigA3 (C)

Fig3.4: Purification of M. tuberculosis LigaseA & its deletion mutants

(A) Purification of MtuLigA: UI-uninduced, !-induced, QS-Q sepharose fraction, HS-Heparin sepharose, BS-Blue sepharose, GF-Gel filtration fraction .

(B) Purification of MtuLigAc: UI-uninduced, !-induced, L-load, Wl-first wash with 50 mM imidazole, W2-second wash with 100 mM imidazole, P-purified fraction and Mr-molecular weight markers (kDa).

(C) Purification of MtuLigAl: Wl-first wash with 50 mM imidazole, W2-second wash with 150 mM imidazole, P-purified fraction. Purification of MtuLigA2 & A3: Wl-first wash with 50 mM imidazole, W2-second wash with 125 mM imidazole, P-purified fraction and Mr-molecular weight markers (kDa).

70

500

400

;:) 300

1 tl § ~ 200 </>

..D <(

,-.,

100

500

400

;:) 300 <( E

tl § ~ 200

</> ..D <(

100

0 5 10 15

Elution Volume (ml)

(A)

MtuLigA

- MtuLigAc

20 25

- MtuLigA I

- MtuLigA2

- MtuLigA3

0 +&~~~--~~~~~~~~~~~----~

0 5 10 15 20 25

Elution Vo lume (ml )

(B)

Fig 3.5: Gel filtration profile of MtuLigA and deletion mutants

(A) Gel filtration profile ofMtuLigA and MtuLigAe-(B) Gel filtration profile ofMtuLigAl , MtuLigA2 and MtuLigA3.

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71

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covalent intermediate (ligase-adenylate) in which AMP is linked via a phosphoamide

bond to the c-amino group of a conserved lysine. In step two, AMP is transferred to the

5' end of the 5' -phosphate-terminated DNA strand to form DNA- adenylate (AppN). In

the step third, ligase catalyzes attack by the 3'-0H of the nick on the DNA-adenylate to

join the two polynucleotide and release AMP. We assayed the ability of the recombinant

constructs to seal a 40mer synthetic duplex DNA substrate containing a single nick

between bases 22 and 23 (Section 2.5.1, Chapter2) (Fig. 3.6). NAD+ and magnesium were

included in the assay mixtures. In case of full-length constructs MtuLigA and MtuLigAc

ligase activity was evinced by conversion of the 5'-32P-labeled 18mer donor strand into an

internally labeled 40mer product visualized by autoradiography of denaturing gel.

Deletional mutant MtuLigAI didn't support any product formation (Fig. 3.7 A).

As discussed above, the initial step in DNA ligation involves formation of a

covalent enzyme-adenylate intermediate. In order to assay adenylyltransferase activity, we

incubated the recombinant LigA and truncated LigA's with e2P-AMP] NAD+ or [a_32P]

A TP and magnesium. While reaction with e2P-AMP] NAD+ resulted in the formation of a 32P-labeled covalent nucleotidyl-protein adduct with all the recombinant forms that co­

migrated with respective polypeptides during SDS-PAGE (Fig. 3.7 B), [a-32P] ATP failed

to react with any of the forms.

Thus, we conclude that Rv3014c codes for a DNA ligase, which uses NAD+ as an

AMP donor. Both native as well as C-terminally his-tagged protein show similar activity

profile thus indicating non-interference of extra residues in the activity efficiency. All the

deletional mutants don't have any ligation activity but have equal adenylyltransferase

activity i.e. they can transfer the AMP moiety ofNAD+ to protein.

This demonstrates that the adenylation domain in itself contains all the essential residues,

which are required in the first step of ligation reaction.

3.2.3 Kinetics of ligation

As discussed above, a synthetic nicked substrate cons1stmg of three

oligonucleotides was employed for Mtu NAD+ -dependent ligase activity assay. A

successful ligation generates an internally labeled 40mer product. Plots of accumulation of

the ligation product versus time showed excellent linearity, allowing to accurately

determine the kinetic constants (Fig. 3.8 A). The enzyme exhibited typical Michaelis­

Menton steady-state kinetics with a Km of~ 88 nM (Fig. 3.8 B, C). The dependence of

72

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3'0H 5'32P

A2 - 22mer A3 - l8mer CCT GGA CAT AGA CTC GTA CCT T AGC TGG ATC ACT GGA CAT

GGA CCT GTA TCT GAG CAT GGA A TCG ACC TAG TGA CCT GTA

AI- 40mer

MtuLigA

CCT GGA CAT AGA CTC GTA CCT T --AGC TGG ATC ACT GGA CAT

GGA CCT GTA TCT GAG CAT GGA A TCG ACC TAG TGA CCT GTA

Denaturing urea PAGE

Autoradiogram

Fig 3.6: Assay procedure of M. tuberculosis Ligase A

A3 (18 mer), A2 (22 mer) and Al (40 mer) are joined together (materials and methods, Section 2.5.1) to prepare an assay substrate having a singly placed nick between 22"d and 23 rd bases of the substrate. Prior to preparation, Al is labeled with 32P-y-ATP. Once the ligations occur, 18mer Al is joined by 22 mer A2 thus trapping the labeled phosphorus in- between. Denaturing urea PAGE leads to separation of A3 (substrate) from newly formed 40 mer (blue) product. Marker dyes, xylene cyanol FF and bromophenol blue are run alongwith to ascertain the position of the oligos in gel.

73

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I 2 3 4 5 6 7 8 9 10

(A)

1 2 3 4 5 6 7 8

-- ~--.,

"'-"'~ - ~- -~~~-

(B)

Fig 3.7: Activity and Adenylation assay ofMtuLi2A & mutants

(A) Activity assay of M. tuberculosis LigaseA and mutants. From left to right- 1: EcoLigA; 2: Control; 3: MtuLigA; 4: Cold treatment; 5: MtuLigAc; 6: Cold treatment; 7: MtuLigAI ; 8: MtuLigA2; 9: MtuLigA3 .

(B) Adenylyltransferase activity of M. tuberculosis LigaseA and mutants. From left to right- Lane I to 4: MtuLigA; MtuLigA I, MtuLigA2; MtuLigA3 respectively with e2P] NAD+. Lane 5 to 8: MtuLigA; MtuLigAl , MtuLigA2; MtuLigA3 respectively with [a-32P] A TP.

74

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LigA nick joining activity on nucleotide cofactor were also tested. MtuLigA exhibits strict . specificity for NAD+ nucleotide cofactor. Nucleotides were omitted from control

reactions. There is no residual activity with any other co factors viz. dA TP, NMN, A TP,

GTP etc. (Fig. 3.9 A). Ligase activity increased with NAD+ concentration up to 5 J..LM. A

Km of 1.46 J..LM was calculated using double reciprocal plot of the data (Fig. 3.9 B).

3.2.3.1 Effect of metal cofactors

Nick ligation required an exogenous divalent cation cofactor. Mg2+ is the preferred

metal ion for MtuLigA. No activity is observed without Mg2+. Optimum concentration for

Mg2+ is 1 0 mM. Increasing the Mg2+ concentration beyond 20 mM leads to increased

inhibition in strand joining. At 40 mM Mg2+, ligation efficiency is 3.5 to 4 times lower

compared to 10 mM Mg2+ (Fig. 3.10 A, B). Mg2+was substituted with alkaline earth metal

Ca2+ and commonly studied period 4 transition metal ions. MtuLigA couldn't use any

other as an alternative metal cofactor. Using Ca2+ as a metal cofactor, MtuLigA were able

to carry out the first 2 steps of ligation reaction i.e. the transfer of AMP to enzyme from

NAD+ and further transfer of it to substrate to form DNA-adenylate intermediate (step 2 in

ligation reaction), however, Ca2+ didn't support the nick closure activity (step 3 in legation

reaction) which led to support ligation or any other intermediate formation. We also

checked the effect of these divalents on enzyme-adenylate formation (E-AMP, step 1 in

ligation reaction). Results show that EpA formation depended on type of divalent cation

cofactor and its concentration. Mg2+ was most optimal of all the cofactors. Optimum EpA

formation occurred in a concentration range of 5 mM to 20 mM. Among other metals,

only Ca2+ supports EpA formation in a narrow concentration range of 5 to 10 mM, the

yield ofEpA at 10 mM Ca2+ was ~5% ofthe optimal value formed with Mg2+.

3.2.3.2 Effects of pH and salt on activity

We checked the activity profile of MtuLigA in the pH range of 6.5 to 9.5. The

optimal pH for MtuLigA was 8.0 with greater than 60% activity observed between 7.5

and 8.5 (Fig. 3.11 A). Activity declined progressively before pH 7.0 and beyond pH 8.5.

MtuLigA activity was tested in two different salts, NaCl and KCl at varying ·

concentrations from 0 to 300 mM. Comparative studies with increasing salt concentrations

shows that MtuLigA has the maximum activity without any salt. It is more sensitive to

inhibition by increasing concentrations of KCl as compared to NaCl with a reduction in

activity by a factor of 30 at 300 mM KCl while same concentration of NaCl reduces the

enzymatic activity by a factor of 2 (Fig. 3.11 B). Decrease in activity is much slower in

NaCl with only marginal loss in activity at initial salt concentrations.

75

0 2

450

400

350 ~

·~ 300 ·= = 250 0 .E 200 .._, > 150

. 100

50

100

0.008

-0.02

4

Time (min)

(A)

200 300

(S] (nM)

(B)

(C)

6 8

400 500

0,03

Fig 3.8: Steady state kinetics of M. tuberculosis LigaseA

Cha ter 3

(A) Time course of ligation reaction containing 5 pmol of ligase and following substrate concentrations: 50 (diamond), 100 (closed square), 200 (triangle) & 400 nM (open circle).

(B) The velocity as function of substrate concentration follows Michaelis-Menten kinetics.

(C) Double reciprocal plot of 1/[V] versus 1/[S]. Sequence of the substrate is depicted in figure 3.6. Kinetic constants were calculated as multiple independent measurements and experimental error is estimated to be 20 %.

76

400

350

.9 300 s 2 250 -,

§ 150 ·g .~ 100 ....l

50

0 ...._ _ __j

None )lAD AlP dA1P G1P C1P U1P

NTP

(A)

400

360

320

~ 280 0 240 ~

---0 200 ......

<.E 160 '-' ;;;.

120

80

40

0 0.0 2.5 5.0 7.5 10.0 12.5 15.0

[NAD] (~L1vi)

(B)

Fig 3.9: Nucleotide cofactor specificity and NAD+ concentration dependence

Cha ter 3

(A) Nucleotide specificity: Reaction mixtures (15 J.tl) containing 50 mM Tris-HCl, pH 8.0, 5 mM DTT, 10 mM MgC12, 2 pmol nicked labeled DNA, 5 pmol ligase and 1 mM NTP or dNTP were incubated for 10 min at 25°C.

(B) NAD concentration dependence: Reaction mixtures (15 J.tl) containing 50 mM Tris-HC1, pH 8.0, 5 mM DTT, 10 mM MgCh, 2 pmo1 nicked DNA, 2 pmol ligaseA and NAD+ as indicated, were incubated for 10 min at 25°C. The extent of ligation was plotted as a function ofNAD+ concentration.

77

--, - _,

Cd2+ Mg2+ NP+ Mn2+ Ca2+ Cu2+

(A)

350

300

-? - 250 -~ --0 -- 200 "0 Mo-2+ -- "' <+:::: 150 ..._, ..... 6

·D 100 co::: OfJ .......

.....:l 50

0

0 10 20 30

Metal con:entration (niM) (B)

Fig 3.10: Divalent cation specificity

Zn2+

40

Cha ter 3

40mer

AppDNA

~pDNA

(A) Assay mixture (15 J.!l) containing 50 mM Tris-HCl, pH 8.0, 5 mM DTT, 50 J.!M NAD+, 2 pmol nicked labeled substrate, 2 pmol ligase and 10 mM divalent cations (Mg, Mn, Ca, Cu, Cd, Zn and Ni) were incubated for 10 min at 25°C. Products were resolved by urea PAGE, autoradiogram of the gel is shown.

(B) Reaction mixtures containing 5 mM DTT, 50 J.!M NAD+, 2 pmol nicked DNA substrate, 2 pmolligase and 50 mM Tris-HCl, pH 8.0 were incubated for 10 min at 25°C at varying Mg2+ concentration ranging from 0 to 40 mM. Extents of ligation were plotted as a function of divalent concentration.

78

Cha ter 3

3.2.4 Deadenylation and DNA binding activity

Deadenylation assays were carried out to ascertain the effect of the C-terminal

deletion on step 2 of the ligation reaction i.e. the ability of the adenylated enzyme to

transfer the bound AMP to nicked substrate. LigaseA and deletion mutants were properly

deadenylated and then adenylated as observed through differential mobility on SDS gel

compared to deadenylated forms. All the four forms have the active site lysine (Section

3.2.1). Only the full-length protein is deadenylated by phosphorylated nicked DNA while

no such activity exists in any of the deletion mutants (Fig. 3.12 A). Quantitative analysis

of the radioactivity in each band (Fig. 3.12 B) reveals that approximately 60 % enzyme

gets deadenylated. Inactivity of MtuLigA1 leads to conclusion that residues present in the

C-terminal BRCT domain play a crucial role in the catalytic transfer of bound AMP to the

5' end of the nicked substrate. Quantitative gel shift assay were also carried-out to check

the relative affinities of enzyme with the nicked and intact dsDNA substrates. Full-length

ligAs viz. MtuLigA and MtuLigAc show strong affinity for nicked duplex DNA substrate

used in the assay. Enzyme shows ~50 times less affinity for intact duplex DNA substrate

with the same composition as nicked one at saturating protein concentrations (Fig. 3.13

A). None of the deletion mutants have any affinity for any form of DNA (Fig. 3.13 B)

suggesting a role for BRCT and helix-hairpin-helix domains.

3.3 Discussion

Sequence alignment studies, activity assays and mutational analysis have

confirmed that Rv3014c is indeed a NAD+ -dependent DNA ligase from Mycobacterium

tuberculosisH37Rv. The clones MtuLigA and MtuLigAc are active. Studies on deletion

mutants have highlighted interesting structure-function features.

In MtuLigA the N-terminal domain is a fully functioning self-adenylation module

as confirmed through adenylyltransferase activity and the C-terminal domain is a fully

functional nicked DNA recognition unit demonstrated by deadenylation and gel mobility

shift assays. Contrary to the results of Thermus filiformis BRCT deletion mutant (Jeon et

al., 2004) which shows ligation activity, deletion of only the BRCT domain resulted in

complete loss of ligation activity in MtuLigA, but the mutant was still able to form an

AMP-ligase intermediate, suggesting that the defects caused by deletion of the entire

BRCT domain occur primarily at steps after enzyme adenylation. Further, sensitive

deadenylation assays and gel mobility shift assays suggest that helix-hairpin-helix domain

79

Cha ter 3

Fig 3.11: Effect of pH & salt on nick-joining activity

(A) Reaction mixtures {15 J.ll) containing 5 rnM DTT, 10 rnM MgCh, 50J.1M NAD+, 2 pmol nicked DNA substrate, 2 pmolligase and 50 rnM Tris-HCl (pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0. 9.5) were incubated for 30 min at 25°C. Extents of ligation were plotted as a function of pH of the buffer.

(B) Similarly, assay was carried out for increasing salt concentration ranging from 0 rnM to 400 rnM. Extent of ligation was plotted as a function of salt concentration.

80

Cha fer 3

+ 1 + 2 + 3 - +4

··~ ••• (A)

1.2

.- 1 -0 ·--~ 0.8 ~ Q)

""0 0.6 ro Q.) ;::.. ·- 0.4 ....... ro

Q) ~

0.2

0 MtuLi2A Mtnl.ioA. 1 MtnLioi\2 MtuLigA3

Protein

(B)

Fig. 3.12: Deadenylation of MtuLieA and mutants with nicked dsDNA

(A) Adenylated MtuLigA and its deletion mutants MtuLigA1, MtuLigA2 and MtuLigA3 (0.1 !lM) in the presence (+) and absence (-) of 5 pmol of nicked DNA. Lane 1, 2, 3 & 4 contains MtuLigA, MtuLigA1, MtuLigA2 & MtuLigA3 respectively. Reactions were incubated at room temperature for four hours prior to analysis on SDS-12% polyacrylamide gels.

(B) Graphical representation of this experiment. The amount of radioactivity in each band was quantified using image master software and then each pair of values were scaled such that the relative adenylation in the absence of nicked duplex DNA was 1.0. (•) Nicked duplex DNA+; (o) nicked duplex DNA-.

81

Cha ter 3

1 2 3 4 5 6 a b c d _e

.... . ~.

c .. ~

···~· '

(A)

A B c D E

• (B)

Fig 3.13: Quantitative band shift assays reveal the relative affinities of MtuLigA and deletional mutants for a nicked and intact duplex DNA substrate of same composition in the absence of magnesium ions. (A) Lane 1 to 6; MtuLigA with nicked assay substrate (Materials and methods).

Protein concentrations were: lane 1, 0 jlg; 2, 3 jlg with no substrate; 3, 3 jlg; 4, 5 jlg; 5, 10 jlg; 6, 15 jlg. Lane a to e; MtuLigA with intact duplex substrate. Protein concentrations were same as in the case with nicked substrate. Reaction mixes were incubated at room temperature for 30 minutes prior to analysis on native 6 % acrylamide gels.

(B) Affinity of MtuLigA and deletion mutants for nicked substrate. Lane A, Control; Lane B, MtuLigA; Lane C, MtuLigA1; Lane D, MtuLigA2; Lane E, MtuLigA3.

82

Cha ter 3

acts in close co-ordination with BRCT domain to play a role in recognizing and binding

DNA substrates which in turn help in execution of step 2 and 3 of ligation reaction. Full­

length ligA shows positive results in both these assays, thus confirming the role of these

domains in M tuberculosis ligA's catalytic action. Step 1 in ligation reaction is achieved

by all the 4 forms of LigA with equal efficiency, indicating the role of conserved motifs

and self-sufficiency of the N-terminal adenylation domain, MtuLigA3 in our studies, in

transfer of AMP from NAD+ to enzyme forming an enzyme-AMP adduct. Mycobacterium

tuberculosis NAD+ -dependent DNA ligase is more specific towards nicked DNA

substrates. Band shift assays confirm that ligA recognize nicked DNA substrates with

more affinity compared to intact dsDNA.

We have determined a Km of- 88 nM for DNA substrate which is similar to that of

E. coli (Modrich et al., 1973) and Thermus ligases (Tong et al., 1999) and 11 fold higher

than that of the Vaccinia virus ATP -dependent ligase (Schiguchi and Shuman, 1997) and

in good agreement with other known NAD+ ligases for such substrate (Tong et al., 2000,)

and 1.4 7 J.!M for NAD+ without any additive. Increasing salt and magnesium leads to low

ligation activity in vitro. It is more sensitive to increasing KCl concentrations as compared

to NaCl. Mg2+ is the preferred metal ion while a profound step 3 arrest and accumulation

of high levels of DNA-adenylate occurs with the inclusion of Ca2+ as a divalent cation

cofactor. Perhaps, at substrate adenylation step, the metal binding site is able to

accomodate Ca2+, thus AppDNA species is observed. However, the subsequent

conformational change may alter the metal-binding site geometry such that it becomes

difficult to accommodate Ca2+ and causes dissociation of the ligase-AppDNA-Ca z+

complex thereby limiting conversion of the intermediate into ligation products.

83