2
Structure Previews How to Build a DNA Unwinding Machine Mark D. Szczelkun 1 and Mark S. Dillingham 1, * 1 DNA:Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, BS8 1TD, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2012.06.006 In this issue of Structure, He and colleagues present the crystal structure of the phage T4 Dda helicase bound to DNA, providing new insights into how this protein efficiently couples single-stranded DNA translocation to DNA strand separation. DNA helicases are motor proteins that separate the DNA duplex into its com- ponent strands in an ATP-dependent manner. Single-stranded DNA is a key intermediate in a wide range of cellular processes including replication, recombi- nation, and repair, and so helicases are ubiquitous, abundant, and often essential proteins for genome maintenance. This diverse group of enzymes encom- passes several distinctive primary struc- ture classes and at least two fundamen- tally different architectures. Moreover, enzymes classified as helicases also in- clude motor proteins that move along dsDNA (or RNA) without unwinding as well as enzymes that remodel nucleopro- tein complexes without processive trans- location. This diversity of function reflects a shared ability to transduce energy from the binding and hydrolysis of ATP to the manipulation and processing of a variety of different nucleic acid or nucleoprotein targets. In the largest two superfamilies (SF1 and SF2) of helicase-like proteins, the motor is formed from tandem RecA domains that bind a single ATP molecule at their interface (Velankar et al., 1999; Figure 1). In bona fide unwinding en- zymes, these core domains bind single- stranded DNA (ssDNA) across their top surface, and cycles of ATP hydrolysis drive conformational changes that move the protein in either the 3 0 -5 0 (class A) or 5 0 -3 0 (class B) direction. Although this motor activity underpins processive DNA unwinding, effective strand separation re- quires additional structural elements that target and distort the incoming duplex. Prominently, many helicases display a ‘‘pin’’ or ‘‘wedge’’ structure located im- mediately ahead of the core motor at the ss-dsDNA junction (Figure 1). This typi- cally engages the final base pair of the duplex and helps to divert the displaced strand. Work from Stephen White’s labo- ratory, presented in this issue of Struc- ture, now provides a new example of such a pin in the Dda helicase and vividly demonstrates its importance for effective DNA unwinding (He et al., 2012). Dda is a model SF1B helicase involved in phage T4 replication and is excep- tionally efficient in coupling ATP hydro- lysis to processive DNA strand separation (Byrd et al., 2012). To understand the structural basis for this robust unwinding, He et al. (2012) co-crystallized Dda with a short ssDNA molecule. The structure reveals the intimate interactions between the helicase core domains and the trans- located DNA strand, which show simi- larities to the related RecD2 helicase (Saikrishnan et al., 2009). However, the Dda structure reveals a new triple stack- ing interaction between a nucleobase and a conserved phenylalanine and pro- line. These amino acid residues are found within a beta-hairpin that is inserted within core domain 1A and which forms an exemplar unwinding pin. Mutagen- esis of the phenylalanine completely uncouples ATP hydrolysis and DNA Figure 1. Examples of DNA Unwinding Pins in Superfamily 1 and 2 Helicases Crystal structures of PcrA (3PJR; Velankar et al., 1999), RecQL1 (2V1X; Pike et al., 2009), Dda (3UPU; He et al., 2012), XPD (4A15; Kuper et al., 2012), and RecBCD (3K70; Singleton et al., 2004) are shown. The helicase core domains 1A and 2A are shown in red and blue respectively, and (where present) nucleotides bound at their interface are shown as black sticks. The pin (or putative pin) structure is shown in mint green and highlighted with an asterisk. In some cases, residues within this structure that are important for coupling hydrolysis to unwinding are shown in stick format. In RecBCD, a second pin structure in the RecD subunit is shown in black and highlighted with a black asterisk. Other domains within each protein are colored light gray and shown as a thin C-alpha ribbon. Where present, DNA is colored yellow, and this always binds across the core domains such that the 3 0 -end is associated with the 1A core domain. The direction of movement of the protein relative to DNA is indicated by arrows and this is opposite for the class A (3 0 -5 0 ) and class B (5 0 -3 0 ) enzymes. Note that the pin is always ‘‘ahead’’ of the ssDNA binding site with respect to translocation direction of the helicase. Structure 20, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1127

How to Build a DNA Unwinding Machine

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Page 1: How to Build a DNA Unwinding Machine

Structure

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How to Build a DNA Unwinding Machine

Mark D. Szczelkun1 and Mark S. Dillingham1,*1DNA:Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, BS8 1TD, UK*Correspondence: [email protected]://dx.doi.org/10.1016/j.str.2012.06.006

In this issue of Structure, He and colleagues present the crystal structure of the phage T4 Dda helicase boundto DNA, providing new insights into how this protein efficiently couples single-stranded DNA translocation toDNA strand separation.

Figure 1. Examples of DNA Unwinding Pins in Superfamily 1 and 2 HelicasesCrystal structures of PcrA (3PJR; Velankar et al., 1999), RecQL1 (2V1X; Pike et al., 2009), Dda (3UPU; Heet al., 2012), XPD (4A15; Kuper et al., 2012), and RecBCD (3K70; Singleton et al., 2004) are shown. Thehelicase core domains 1A and 2A are shown in red and blue respectively, and (where present) nucleotidesbound at their interface are shown as black sticks. The pin (or putative pin) structure is shown inmint greenand highlighted with an asterisk. In some cases, residues within this structure that are important forcoupling hydrolysis to unwinding are shown in stick format. In RecBCD, a second pin structure in theRecD subunit is shown in black and highlighted with a black asterisk. Other domains within each proteinare colored light gray and shown as a thin C-alpha ribbon. Where present, DNA is colored yellow, and thisalways binds across the core domains such that the 30-end is associated with the 1A core domain. Thedirection of movement of the protein relative to DNA is indicated by arrows and this is opposite for theclass A (30-50) and class B (50-30) enzymes. Note that the pin is always ‘‘ahead’’ of the ssDNA bindingsite with respect to translocation direction of the helicase.

DNA helicases are motor proteins that

separate the DNA duplex into its com-

ponent strands in an ATP-dependent

manner. Single-stranded DNA is a key

intermediate in a wide range of cellular

processes including replication, recombi-

nation, and repair, and so helicases are

ubiquitous, abundant, and often essential

proteins for genome maintenance. This

diverse group of enzymes encom-

passes several distinctive primary struc-

ture classes and at least two fundamen-

tally different architectures. Moreover,

enzymes classified as helicases also in-

clude motor proteins that move along

dsDNA (or RNA) without unwinding as

well as enzymes that remodel nucleopro-

tein complexes without processive trans-

location. This diversity of function reflects

a shared ability to transduce energy from

the binding and hydrolysis of ATP to the

manipulation and processing of a variety

of different nucleic acid or nucleoprotein

targets.

In the largest two superfamilies (SF1

and SF2) of helicase-like proteins, the

motor is formed from tandem RecA

domains that bind a single ATP molecule

at their interface (Velankar et al., 1999;

Figure 1). In bona fide unwinding en-

zymes, these core domains bind single-

stranded DNA (ssDNA) across their top

surface, and cycles of ATP hydrolysis

drive conformational changes that move

the protein in either the 30-50 (class A) or

50-30 (class B) direction. Although this

motor activity underpins processive DNA

unwinding, effective strand separation re-

quires additional structural elements that

target and distort the incoming duplex.

Prominently, many helicases display a

‘‘pin’’ or ‘‘wedge’’ structure located im-

mediately ahead of the core motor at the

ss-dsDNA junction (Figure 1). This typi-

cally engages the final base pair of the

duplex and helps to divert the displaced

strand. Work from Stephen White’s labo-

ratory, presented in this issue of Struc-

ture, now provides a new example of

such a pin in the Dda helicase and vividly

demonstrates its importance for effective

DNA unwinding (He et al., 2012).

Dda is a model SF1B helicase involved

in phage T4 replication and is excep-

tionally efficient in coupling ATP hydro-

lysis to processive DNA strand separation

(Byrd et al., 2012). To understand the

structural basis for this robust unwinding,

He et al. (2012) co-crystallized Dda with

a short ssDNA molecule. The structure

Structure 20, July 3, 2012 ª

reveals the intimate interactions between

the helicase core domains and the trans-

located DNA strand, which show simi-

larities to the related RecD2 helicase

(Saikrishnan et al., 2009). However, the

Dda structure reveals a new triple stack-

ing interaction between a nucleobase

and a conserved phenylalanine and pro-

line. These amino acid residues are found

within a beta-hairpin that is inserted

within core domain 1A and which forms

an exemplar unwinding pin. Mutagen-

esis of the phenylalanine completely

uncouples ATP hydrolysis and DNA

2012 Elsevier Ltd All rights reserved 1127

Page 2: How to Build a DNA Unwinding Machine

Structure

Previews

translocation from DNA unwinding. Fur-

thermore, this pin is structurally sup-

ported by interactions with a ‘‘tower’’

domain that locks it rigidly in place,

creating an arch throughwhich the ssDNA

passes onto the core motor. Remarkably,

simply destabilizing the pin by muta-

tion of its interface with the tower domain

caused substantial defects in DNA

unwinding.

Unwinding pins have been observed

in many other helicases (Figure 1), but

their importance for DNA unwinding may

vary, even in closely-related systems.

The RecQ helicases provide an inter-

esting example, as the size and apparent

importance of the pin varies between

family members. In human RecQL1, a

conserved tyrosine residue at the tip of

the pin is critical for coupling ATP hydro-

lysis to unwinding, whereas E. coli RecQ

is rather tolerant of similar mutations

(Pike et al., 2009). The pin can also adopt

architectures other than the common

beta-hairpin. In the SF2B helicase XPD,

a duplex separation wedge is thought to

1128 Structure 20, July 3, 2012 ª2012 Elsevi

be formed by an iron-sulfur domain asso-

ciated with the 1A core domain and

disruption of the cluster by mutagenesis

uncouples DNA unwinding from ATP

hydrolysis (Pugh et al., 2008; Kuper

et al., 2012). In the bipolar helicase

RecBCD, a putative pin is not found in

either of the RecB or RecD helicase

subunits, but is instead contributed by

a short loop in the RecC polypeptide,

which stacks a methionine dyad directly

against the terminal base pair of the

duplex (Singleton et al., 2004). Curiously,

a secondary pin element is present in

RecD, despite the fact that DNA arrives

at this motor as pre-formed ssDNA.

Finally, pin and wedge domains are not

only a feature of DNA unwinding en-

zymes but also of motors that remodel

branched DNA structures such as RecG

and RuvAB. While the pin performs a

similar role, in that it re-directs the duplex

strands onto separate paths, the ssDNA

immediately anneals with a new partner

strand to promote Holliday junction or

replication fork remodeling.

er Ltd All rights reserved

REFERENCES

Byrd, A.K., Matlock, D.L., Bagchi, D., Aarattutho-diyil, S., Harrison, D., Croquette, V., and Raney,K.D. (2012). J. Mol. Biol. 420, 141–154.

He, X., Byrd, A.K., Yun, M.-K., Pemble, C.W., IV,Harrison, D., Yeruva, L., Dahl, C., Kreuzer, K.N.,Raney, K.D., and White, S.W. (2012). Structure20, this issue, 1189–1200.

Kuper, J., Wolski, S.C., Michels, G., and Kisker, C.(2012). EMBO J. 31, 494–502.

Pike, A.C., Shrestha, B., Popuri, V., Burgess-Brown, N., Muzzolini, L., Costantini, S., Vindigni,A., and Gileadi, O. (2009). Proc. Natl. Acad. Sci.USA 106, 1039–1044.

Pugh, R.A., Honda, M., Leesley, H., Thomas, A.,Lin, Y., Nilges, M.J., Cann, I.K., and Spies, M.(2008). J. Biol. Chem. 283, 1732–1743.

Saikrishnan, K., Powell, B., Cook, N.J., Webb,M.R., and Wigley, D.B. (2009). Cell 137, 849–859.

Singleton, M.R., Dillingham, M.S., Gaudier, M.,Kowalczykowski, S.C., and Wigley, D.B. (2004).Nature 432, 187–193.

Velankar, S.S., Soultanas, P., Dillingham, M.S.,Subramanya, H.S., and Wigley, D.B. (1999). Cell97, 75–84.

Regulated Proteolysis as a Forceto Control the Cell Cycle

Jodi L. Camberg1,* and Sue Wickner1,*1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA*Correspondence: [email protected] (J.L.C.), [email protected] (S.W.)http://dx.doi.org/10.1016/j.str.2012.06.004

In this issue of Structure, Rood and colleagues report that substrate architecture is a key factor in promotingthe complete and processive degradation of the Caulobacter cell cycle regulator PdeA by the proteaseClpXP. This investigation highlights the important role that the adaptor protein CpdR serves in regulatingpresentation of PdeA to ClpXP.

The degradation of cell cycle regulators

during the cell division cycle of Caulo-

bacter crescentus is critical for controlling

the timing of events such as DNA replica-

tion and cell division (Curtis and Brun,

2010). A driving force for the programmed

transition from G1 to S-phase is ClpXP, a

two-component ATP-dependent pro-

tease. Through degradation of the cell

cycle regulator, CtrA, and the phosphodi-

esterase PdeA, ClpXP orchestrates entry

into S-phase (Chien et al., 2007; Jenal

and Fuchs, 1998).

In this issue of Structure, Rood et al.

(2012) describe structural organization

and examine regulated degradation of

PdeA. PdeA antagonizes the activity of

DgcB, a diguanylate cyclase (Abel et al.,

2011). When PdeA is degraded by ClpXP,

DgcB is left unchecked, creating a situa-

tion that causes an upshift in cyclic-

di-GMP and leads to CtrA degradation.

PdeA degradation by ClpXP requires an

adaptor protein, CpdR, which is also

a response regulator. CpdR functions in

the traditional role of a substrate-specific

adaptor protein, facilitating the degrada-

tion of PdeA.

ClpXP is a member of the protein

quality control machinery with structural

organization and a mechanism of degra-

dation similar to the eukaryotic 26S pro-

teasome (Baker and Sauer, 2012; Ortega