Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass

Molecular Cell

Article Human DNA Polymerase k Encircles DNA: Implications for Mismatch Extension and Lesion Bypass Samer Lone,1,3 Sharon A. Townson,1,3 Sacha N. Uljon,1,3,4 Robert E. Johnson,2 Amrita Brahma,2 Deepak T. Nair,1 Satya Prakash,2 Louise Prakash,2 and Aneel K. Aggarwal1,* 1 Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, NY 10029, USA 2 Sealy Center for Molecular Science, University of Texas Medical Branch, 6.014 Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555, USA 3 These authors contributed equally to this work. 4 Present address: Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02155, USA. *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.01.018

SUMMARY

Human DNA polymerase k (Pol k) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol k catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique ‘‘N-clasp’’ at the N terminus of Pol k, which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol k’s ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase. INTRODUCTION External and internal DNA-damaging agents continually threaten the integrity of genetic material in cells. Although organisms have evolved a variety of repair mechanisms to remove the resulting lesions, some lesions escape repair and block the replication machinery. The recently discovered Y family DNA polymerases permit the continuity of the replication fork by allowing replication through such DNA lesions (Prakash et al., 2005). Humans have four Y family polymerases—Pol h, Pol i, Pol k, and Rev1—each with a unique DNA-damage bypass and fidelity profile. Pol h, for example, is unique in its ability to replicate

through an ultraviolet (UV)-induced cis-syn thyminethymine (T-T) dimer by inserting two As opposite the two Ts of the dimer with the same efficiency and accuracy as opposite undamaged Ts (Johnson et al., 1999b, 2000c; Washington et al., 2000, 2003). Because of the involvement of Pol h in promoting error-free replication through cyclobutane pyrimidine dimers, its inactivation in humans causes the variant form of xeroderma pigmentosum, a genetic disorder characterized by a greatly enhanced predisposition to sun-induced skin cancers (Johnson et al., 1999a; Masutani et al., 1999). Pol i, on the other hand, is unable to replicate through a cis-syn T-T dimer (Haracska et al., 2001; Johnson et al., 2000b) but can proficiently incorporate nucleotides opposite N2-adducted guanines, such as 1,N2-propano-20 deoxyguanosine (g-HOPdG) and trans-4-hydroxy-2-noenal-deoxyguanosine (HNE-dG) (Washington et al., 2004; Wolfle et al., 2006) that result from the peroxidation of lipids, and which are present in the DNA of human tissues at considerable levels. In all, Y family polymerases in eukaryotes display a large degree of functional divergence, rendering them highly specialized for specific roles in lesion bypass (Prakash et al., 2005). Pol k is specialized for the extension step of lesion bypass. On undamaged DNAs, for example, Pol k misincorporates nucleotides with a frequency of 103–104 (Johnson et al., 2000a), whereas it extends mispaired termini almost two orders of magnitude more efficiently with a frequency of 101–102 (Washington et al., 2002). Also, Pol k is unable to insert nucleotides opposite the 30 T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 30 T of the dimer by another DNA polymerase (Johnson et al., 2000a; Washington et al., 2002). The g-HOPdG and HNE-dG adducts also present a strong block to nucleotide incorporation by Pol k, but the polymerase carries out efficient extension from a C nucleotide incorporated opposite these adducts by another polymerase, such as Pol i (Washington et al., 2004; Wolfle et al., 2006). Together, these results are part of growing evidence that lesion bypass in

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Figure 1. Requirement of N Terminus for Pol k Function (A) Schematic alignment of human Pol k with E. coli DinB (Pol IV) and S. solfataricus Dpo4. Regions corresponding to motifs I–V, common to all Y family polymerases, are shown in different colors. Regions that form the architectural palm, fingers, and thumb domains are shown in red, yellow, and orange, respectively. The x, y, and z motifs in Pol k are shared among all members of the DinB subfamily. The N-terminal region of 75 residues is present in Pol k but not in DinB or Dpo4. (B) DNA synthesis from matched and mismatched primer termini opposite from template G. Full-length Pol k1–870 (0.5 nM) or truncated Pol k1–526 (0.5 nM), Pol k19–526 (0.5 nM), or Pol k68–526 (50 nM) were incubated with 10 nM DNA substrate for 5 min at 37 C under the standard polymerase reaction conditions that contained 25 mM of each of the four dNTPs. In the DNA sequence shown on top, N in the primer refers to any of the four dNTPs. (C) DNA synthesis from an A paired with an undamaged T or the 30 T of a cis-syn T-T dimer (CPD). Reaction conditions were the same as in (B). A portion of the DNA substrate is shown on top, and the identities of the nucleotide in the template and the primer at the template-primer junction are indicated below each lane.

eukaryotes often requires the sequential action of two polymerases, an ‘‘inserter’’ and an ‘‘extender.’’ The inserter is efficient at insertion of an incoming nucleotide across from the lesion, and the extender, such as Pol k or Pol z (a B family polymerase), is recruited to add bases downstream of the lesion (Prakash et al., 2005). Pol k is the only human Y family polymerase with homologs in prokaryotes and archaea, including DinB (Pol IV) in Escherichia coli and Dbh and Dpo4 in Sufolobus solfataricus (Figure 1A), and it shares with them a tendency to generate frameshift mutations (Kim et al., 1997; Kobayashi et al., 2002; Kokoska et al., 2002; Ogi et al., 1999; Ohashi et al., 2000). However, the mechanism of frameshift mutagenesis differs between Pol k and its prokaryotic and archaeal homologs (Wolfle et al., 2003). Pol IV and Dpo4 are also much less efficient at extending mispaired termini than Pol k (Kobayashi et al., 2002; Trincao et al., 2004). Furthermore, the amino acid (aa) sequence of Pol k is set apart from Pol IV and Dpo4 (and other Y family members) by an extension at the N terminus of 75 amino acids (Figure 1A). The N-terminal extension is indispensable for Pol k activity and is conserved only among eukaryotic Pol k proteins (Uljon et al., 2004).

Structural information on Pol k is currently limited to the catalytic core of human apo Pol k (aa 68–526) (Uljon et al., 2004). Although this structure was determined in the absence of DNA and lacked the first 67 amino acids (important for activity), it did reveal architecture similar to that of other Y family polymerases with palm, fingers, and thumb domains in the shape of the right hand, and an additional domain, termed the PAD (for polymerase-associated domain) or ‘‘little finger’’ (Ling et al., 2001; Nair et al., 2004, 2005b; Silvian et al., 2001; Trincao et al., 2001; Zhou et al., 2001). However, compared to other Y family polymerases, the Pol k thumb domain was found to be topologically different, and the PAD was observed tucked under the palm domain and not juxtaposed close to the fingers domain. We present here a crystal structure of the human Pol k catalytic core (aa 19–526) bound to a template-primer and an incoming nucleotide. The structure reveals almost complete encirclement of the DNA and a constrained active-site cleft that can accommodate only a single Watson-Crick base pair. These and other observations help explain Pol k’s specialized role in mismatch extension and its role in extending opposite from DNA lesions.

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Table 1. Steady-State Kinetic Parameters for Extension of Matched and Mismatched Primer Termini by Full-Length and Truncated Pol k Proteins Template:Primer Base Pair

Pol k 1–870

G$C

G$T

G$G

G$A

T$G

T*$A

9.8 ± 0.08

Km (mM)

Efficiency kcat/Km

Efficiency Relative to Pol k1–870

2.8 ± 0.09

3.5

1

1–526

20.3 ± 1.0

4.8 ± 1.0

4.2

1.4

19–526

36.5 ± 0.5

4.2 ± 0.09

8.7

2.5 2

1–870

6.8 ± 0.2

107 ± 9

6.4 3 10

1

1–526

16.8 ± 0.6

104 ± 12

0.16

2.5

19–526

10.5 ± 1.1

681 ± 205

1.5 3 102 2

0.23

1–870

3.5 ± 0.3

144 ± 29

2.4 3 10

1

1–526

10.1 ± 1.1

233 ± 65

4.3 3 102

1.8

3

19–526

2.0 ± 0.3

581 ± 275

3.4 3 10

0.14

1–870

4.3 ± 0.3

175 ± 32

2.5 3 102

1

180 ± 14

2

1–526

T$A

kcat (min1)

12.8 ± 0.3

7.1 3 10

3

2.8

19–526

3.2 ± 0.1

337 ± 50

9.5 3 10

0.4

1–870

12.8 ± 0.3

1.2 ± 0.1

10.7

1

1–526

37.6 ± 1.2

1.8 ± 0.2

20.9

2

19–526

12.8 ± 0.2

1–870

3.5 ± 0.2

11.7 ± 2.0

1–526

6.0 ± 0.6

5.8 ± 2.4

0.4 ± 0.04

32

3

0.3

1

1

3.3

19–526

1.4 ± 0.1

3.8 ± 1.3

0.4

1.3

1–870

11.3 ± 0.3

14.2 ± 0.8

0.8

1

1–526

21.1 ± 0.5

25.9 ± 3.0

0.8

1

19–526

11.4 ± 1.3

18 ± 1.3

0.6

0.75

0

T* indicates the 3 T of the cis-syn T-T dimer. Full length, aa1–870.

RESULTS Requirement of the N Terminus of Pol k for DNA Synthesis from Matched and Mismatched Primer Termini To determine the contribution that the N terminus of Pol k makes to its proficiency for mismatch extension and for extension opposite from DNA lesions, we compared the abilities of full-length Pol k (1–870) with truncated Pol k (1–526), Pol k (19–526), and Pol k (68–526) for the extension of matched and mismatched primer termini opposite from template G. As is shown in Figure 1B, Pol k1–526 is as active in DNA synthesis from matched and mismatched primer termini as the full-length protein, whereas for Pol k19–526 the proficiency for mismatch extension is somewhat reduced but the ability to extend from matched primer terminus was not affected. In Pol k68–526, on the other hand, the DNA polymerase activity is almost abrogated, since even with 100-fold higher amounts than we used for the other three Pol k proteins, the extension of matched G$C primer terminus was greatly diminished and extension from mismatched primer termini was abolished.

Although Pol k is unable to insert a nucleotide opposite the 30 T of a cis-syn T-T dimer, it proficiently extends from an A opposite this site. To examine the contribution of the N terminus to extension opposite from DNA lesions, we compared the abilities of N-terminally deleted Pol k proteins for extension from an A placed opposite the 30 T of the T-T dimer. Whereas Pol k1–526 and Pol k19–526 were as effective in extending from this primer terminus as the full-length protein, Pol k68–526 was unable to carry out this extension reaction and this protein was also severely impaired in its ability to extend from an A opposite the undamaged T template (Figure 1C). To obtain a quantitative estimation of extension efficiencies of various Pol k proteins, we carried out steady-state kinetic analyses to determine the catalytic efficiencies (kcat/Km) for extension from matched and mismatched primer termini and for extension opposite from a T-T dimer. As shown in Table 1, compared to full-length protein, the efficiencies of extension from matched and mismatched primer termini opposite from template G were the same or somewhat higher in the Pol k1–526 protein, whereas in the Pol k19–526 protein, the mismatch extension

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proficiency opposite from template G was somewhat reduced, and extension from the matched G$C primer terminus was somewhat better. The efficiencies for extension from an A or from a G opposite template T were also about the same in Pol k19–526 as in full-length Pol k or in Pol k1–526, and all three proteins were equally proficient in extending from the A placed opposite the 30 T of a T-T dimer. We conclude from these observations that there is an indispensable role of the Pol k N terminus for DNA synthesis and mismatch extension. In particular, they indicate that residues 19–68 are required for synthesis from both matched and mismatched primer termini and that the first 18 residues contribute additionally to the mismatch extension proficiency of Pol k. Structure Determination We crystallized the Pol k19–526 catalytic core in ternary complex with a 13 nt/18 nt primer/template and incoming dTTP. The cocrystals diffract to 3.05 A˚ resolution with synchrotron radiation (Advanced Photon Source, or APS), and there are two ternary complexes (A and B) in the crystallographic asymmetric unit (Table 2). The structure was determined by molecular replacement (MR) using the apo Pol k68–526 structure (without the PAD) as a search model. For ternary complex A, the final model consists of residues 33–224 and 282–517 of Pol k, nucleotides 3– 15 of the template, nucleotides 4–13 of the primer, incoming dTTP, and an Mg2+ ion. For ternary complex B, the final model consists of residues 21–224 and 282–517 of Pol k, nucleotides 3–17 of the template, nucleotides 2–13 of the primer, incoming dTTP, and an Mg2+ ion. The two ternary complexes are very similar in structure, though complex B is more complete and complex A is better ordered. We describe below the structure of complex B and refer to complex A as needed. Overall Arrangement Pol k encircles DNA—wherein residues 21–74 at the N terminus delineate an ‘‘N-clasp’’ subdomain that augments the conventional right-handed grip on the template-primer by the palm, fingers, and thumb domains, and the PAD (Figure 2). The palm and fingers domains interact primarily with the replicative end of the template-primer, wherein the palm (aa 101–109 and 171–338) carries the activesite residues (D107, D198, and E199) that catalyze the nucleotidyl transfer reaction, while the fingers domain (aa 110–170) drapes over the nascent base pair in the active site formed between templating A and incoming dTTP. The thumb and the PAD straddle the duplex portion of the template-primer, connected by a long linker that cradles one side of the DNA. The thumb (aa 79–100 and 339–401) skims the minor groove surface, while the PAD (aa 401–518) anchors in the major groove. The N-clasp bears some structural resemblance to an ‘‘N-digit’’ in Rev1 (Nair et al., 2005b), but the functions of these two N-terminal subdomains are different. In Rev1, the N-digit stems from the palm domain and specifies the identity of the incoming nucleotide. In Pol k, the N-clasp (aa 21–74)

Table 2. Crystallographic Data Collection, Phasing, and Refinement Statistics Data Collection Wavelength (A˚)

Native 2

Resolution (A˚)

3.05

1.0

Number of measured reflections

185,470

Number of unique reflections

37,424

Data coverage (%)

99.8 (99.7)

Rmerge (%)a,b

4.4 (35.0)

I/s

23.6 (3.6)

Refinement Statistics Resolution range (A˚)

50–3.05

Reflections

35,239

Rcryst (%)c

23.1

Rfree (%)

d

29.4

Nonhydrogen atoms Protein

6,500

DNA

1008

dTTP

58

Mg2+

2

Water

21

Average B factors (A˚2) Protein A

52.4

DNA A

68.4

dTTP A

55.5

Mg2+ A

51.8

Protein B

67.3

DNA B

75.9

dTTP B

53.5

Mg2+ B

56.5

Rms Deviations Bonds (A˚)

0.0078

Angles ( )

1.38

Ramachandran plot quality

Mol A

Mol B

Most favored (%)

85.3

80.2

Additional allowed (%)

13.5

17.6

Generously allowed (%)

2.3

2.2

Disallowed (%)

0.0

0.0

a

Values for shells are given in parentheses. PoutermostP b Rmerge = jI  j / I, where I is the integrated intensity of a given intensity. P P c Rcryst = jjFobservedj  jFcalculatedjj / jFobservedj. d Rfree was calculated using 10% random data omitted from the refinement.

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directly in the major groove in the ternary complex: a movement of 50 A˚ between the two positions. The PAD makes the majority of DNA contacts that stabilize the Pol k ternary complex. The N-clasp may only become ordered in the presence of DNA. Interestingly, the only Pol k construct that we have been able to crystallize in the absence of DNA is Pol k68–526 that lacks most of the N-clasp; this may be because the N-clasp is disordered in the absence of DNA and thus hinders crystallization.

Figure 2. Pol k Encircles DNA (A) Ribbon diagram representing the overall structure of the ternary complex. The palm, fingers, and thumb domains and the PAD are shown in light blue, yellow, orange, and green, respectively. The Nclasp subdomain, unique to Pol k, is highlighted in dark blue. DNA is represented in gray; template dA and incoming dTTP are in red, and a putative Mg2+ ion is in blue. (B) A view of the ternary complex looking down the DNA helix to show encirclement of the DNA by the N-clasp.

extends from the thumb domain and gives the visual impression of a ‘‘lever’’ that swivels from the thumb to the PAD side of the DNA (Figure 2). Pol k is the only Y family DNA polymerase for which structures are now available for both the apo enzyme and the ternary complex. Although the apo enzyme structure lacked the N-clasp, its comparison to Pol k in the ternary complex (with the N-clasp) suggests a large movement of the PAD (Figure 3). Thus, whereas the PAD tucks under and behind the palm domain in the apo enzyme (distant from the DNA-binding surface), it docks

Watson-Crick Base-Pairing and Nucleotide Incorporation The base-pairing in the active-site cleft is Watson-Crick, wherein two hydrogen bonds are established between templating A and incoming dTTP (Figure 4 and Figure S1). The dTTP triphosphate moiety travels between the fingers and palm domains, making hydrogen bonds with Y141 and R144 from the fingers domain and K328 from the palm domain (Figure 4A). Y141, R144, and K328 are conserved in all Y family polymerases, and mutation of the analogous residues in yeast Pol h (Y64, R67, and K159) diminishes the nucleotide incorporation ability of the polymerase (Johnson et al., 2003). The dTTP sugar packs against the aromatic ring of Y112, which is conserved in all Y family polymerases as either a tyrosine or a phenylalanine and has been postulated as a ‘‘steric gate’’ for the exclusion of ribonucleotides (DeLucia et al., 2003). The catalytic residues D107, D198, and E199 are clustered between the triphosphate moiety and the primer terminus (Figure 4A). An Mg2+ ion occupies a position corresponding to ‘‘metal B’’ in replicative polymerases (Doublie et al., 1998; Li et al., 1998; Steitz, 1999) (Figure 4C). The Mg2+ is assigned as a metal ion (rather than as a water molecule) based on its octahedral coordination and short ligation distances. The ion is coordinated in the basal octahedral plane by the unesterified oxygens of dTTP b- and g-phosphates and the carboxylates of D107 and D198 (Figure 4C). The octahedral coordination sphere is completed by oxygens at apical positions, coming from a-phosphate and the main-chain carbonyl of M108. Surprisingly, there is no density for an Mg2+ ion at a position analogous to ‘‘metal A’’ in replicative polymerases or in Y family polymerases Pol i (Nair et al., 2005a) and Rev1 (Nair et al., 2005b). (Typically, metal A is coordinated by the a-phosphate of the incoming nucleotide, the putative primer 30 OH, the carboxylates of active-site residues, and water molecules.) There is, however, density for a water molecule, located 2 A˚ from the site normally occupied by metal A in replicative polymerases. The N-Clasp Locks the Polymerase around the DNA The N-clasp is composed of a loose U-shaped tether at the N terminus followed by a short a helix (aN1) and a long a helix (aN2)—in the shape of an ‘‘L’’ (Figures 2 and 3). aN2 is the dominant feature of the N-clasp that traverses the template-primer at an 45 angle to the DNA axis. The C terminus of aN2 joins the thumb (and also packs against it), while its N terminus makes contacts

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with the PAD. Approximately 834 A˚2 of solvent-accessible surface area is buried between aN2 and the thumb, due mostly to nonpolar interactions between V60, I64, M67, and M68 emanating from the N-clasp and V360, M364, A367, and L368 from the thumb. At the other end of helix aN2, F49 and N52 are involved in van der Waals contacts with F465 on the PAD, burying 248 A˚2 of solvent-accessible surface area over this region. aN1 interacts mainly with the fingers domain, wherein I36 and I39 (built as alanines in complex B) and A43 pack against F155, I156, V160, and R159 on the fingers domain (with 344 A˚2 of solvent-accessible surface area buried). The U-shaped tether reaches across the junction between the fingers and palm domains, and it partially covers the dTTP-binding site. The N-clasp appears to ‘‘lock’’ the thumb, fingers, and palm domains and the PAD of Pol k around the DNA. Approximately 437 A˚2 of solvent-accessible surface area is buried between aN2 and the duplex portion of template-primer, wherein K56 and R63 partially penetrate the major and minor grooves, respectively, but most of the other residues only graze the DNA surface. The most intimate contacts between aN2 and the DNA are with the two unpaired C nucleotides on the 50 side of the templating base. Both of these nucleotides lie outside of the activesite cleft, where the base of the first unpaired C is sandwiched between F49 on aN2 and P153 on the fingers domain and the base of the second unpaired C makes van der Waals contacts with residues at the N terminus of aN2 (Figures 4A and 4B). Helix aN1 makes only few direct contacts with DNA: mainly some van der Waals interactions and a possible hydrogen bond between T44 and the first unpaired template C. To verify the role of the N-clasp in DNA binding, we examined a set of N-terminally deleted Pol k proteins for their binding to a fluorescein-labeled 13 nt/18 nt primer/template using fluorescence anisotropy. As shown in Figure 5, as the first 18 residues and then the N-clasp are progressively deleted there is a concomitant decrease in DNA binding. Thus, whereas Pol k1–526 containing the entire N terminus binds the template-primer with a KD of 100 nM, Pol k19–526 (containing the full N-clasp but lacking the first 18 residues) binds the template-primer with a KD of 170 nM, and Pol k37–526 (with a portion of helix aN1 deleted), Pol k47–526 (with helix aN1 and portion of helix aN2 deleted), and Pol k68–526 (with most of helix aN2 deleted) bind the template-primer with KDs of 340 nM, 410 nM, and 730 nM, respectively. Together, these data suggest that the N-clasp enhances the ability of Pol k to grip the DNA and that the first 18 residues additionally contribute to the proficiency of DNA binding by Pol k.

A Constricted Active-Site Cleft The Pol k fingers domain is smaller than that of yeast Pol h or archaea Dpo4 lacking, for example, the strands analogous to b5 and b6 in Pol h and the large loop between strands b2 and b3 in Dpo4 (Figure 3C). The Pol k activesite cleft is constricted, however, by the fingers domain impinging on templating A. In particular, M135 (emanating from the fingers domain) bears down on the templating base, preventing the next 50 nucleotide from stacking above it (Figures 4A and 4B). Consequently, only templating A is held in the active site, whereas the rest of the 50 unpaired template strand is directed out of the active-site cleft (and is stabilized via interactions with the N-clasp— described above). The inability of Pol k to insert a nucleotide opposite the 30 T of a cis-syn T-T dimer appears to be the result of this constricted active-site cleft that is unable to accommodate two unpaired nucleotides. Accordingly, when we model the 30 T of a cis-syn T-T dimer at the templating position in the Pol k active-site cleft, the 50 T of the T-T dimer (covalently linked to the 30 T by a cyclobutane ring) overlaps with both M135 from the fingers domain and F49 from the N-clasp (Figure 6A). Interestingly, a superposition of the yeast apo Pol h structure onto that of Pol k suggests that the fingers domain is farther away from the templating base in Pol h than in Pol k, thereby allowing both Ts of the T-T dimer to be accommodated in the Pol h active-site cleft. The Pol h active-site cleft is also widened by the substitution of smaller S58 in place of M135 in Pol k, and by the lack of an N-clasp (next to the active-site cleft). Extension from DNA Lesions Following the insertion of a nucleotide opposite a lesion, the lesion base pair is translocated along the templateprimer from the ‘‘insertion’’ T0-P0 to the ‘‘postinsertion’’ T1-P1 position (where T and P refer to template and primer strands, respectively, and the subscripts refer to the number of base pairs from the templating base position). Pol k is better at extending from DNA lesions than it is in inserting nucleotides opposite the lesions. For example, although Pol k is unable to insert nucleotides opposite the 30 T of a cis-syn T-T dimer, it can efficiently extend from a nucleotide inserted opposite the 30 T of the dimer by another polymerase. Accordingly, when we model the 50 T of a cis-syn T-T dimer at the templating T0 position in the active-site cleft, neither the fingers domain nor the N-clasp interferes with the binding of the template. The ‘‘preinsertion’’ nucleotide (at T1 position) in this case can be kinked and accommodated outside of the active-site cleft, between the N-clasp and the fingers domain.

Figure 3. Conformational Changes in Pol k and Its Relation to Other Y Family DNA Polymerases (A) Conformational changes in Pol k upon complex formation. On binding DNA and incoming dNTP, the PAD domain swings 50 A˚ toward the major groove of the DNA. In addition, the N-clasp appears to undergo a disorder-to-order transition—locking Pol k around the DNA. (B) Surface diagrams comparing Pol k to the structures of several Y family polymerases. The colors of the domains coincide with those used in Figure 2, with the N-digit from Rev1 also highlighted in dark blue. (C) The secondary structure and domain topology of Pol k.

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Figure 4. The Pol k Active-Site Cleft (A) A close-up view of the Pol k active-site region. The fingers, palm, and thumb domains and the PAD are shown in yellow, light blue, orange, and green, respectively. The N-clasp is colored dark blue, with the U-shaped tether omitted for clarity. The DNA is colored gray, the template dA and incoming dTTP are in red, and the putative Mg2+ ion is shown in blue. Highlighted and labeled are the catalytic residues (D107, D198, and E199) and the residues apposed close to incoming dTTP (F111, Y112, T138, Y141, R144, and K328), template dA (M135, A151, K461, F465, and R507), and the first unpaired template dC (F49, S134, P153, and F155). (B) Close-up view of the nascent base pair fitting against the Pol k molecular surface. Several residues apposed to the templating dA and unpaired dC base are shown in gray and labeled on the molecular surface. (C) Close-up view of metal ion coordination in the active site. On the left is shown a simulated annealing Fo  Fc omit map (contoured at 3.0 s; 3.05 A˚ resolution), showing a single Mg2+ ion in the active site. Highlighted are the catalytic residues (D107, D198, and E199) and M108. The incoming dTTP is

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Figure 5. DNA-Binding Affinities of NTerminally Deleted Pol k Proteins (A) Schematic representation of N-terminal deletions generated in Pol k. Amino acid residues contained in each protein are indicated on the left. Dissociation constant (KD) for each construct is indicated on the right. (B) Binding curves for the various N-terminally deleted Pol k constructs. The fraction of DNA bound is plotted versus Pol k concentration in order to determine the dissociation constants. Filled circles are for Pol k1–526, open circles for Pol k19–526, filled-in triangles for Pol k37–526, open triangles for Pol k47–526, and filled-in squares for Pol k68–526.

The ability of Pol k to extend proficiently from minor groove adducts such as g-HOPdG and HNE-dG may derive from the fact that the minor groove edge of the postinsertion T1 nucleotide (opposite the primer terminus) is open to solvent and relatively unobstructed by the polymerase. The nearest Pol k residues in the plane of the T1 base, namely R175 and L197 on the palm, are located >10 A˚ from the minor groove edge of the postinsertion T1 nucleotide. As such, the ring-open form of HNE-dG adduct is unobstructed when modeled at T1 position in the Pol k ternary complex but overlaps with residues Y39 and F125 in the Pol i ternary complex (Figure 6B).

DISCUSSION Pol k is the most faithful of all Y family polymerases, misincorporating nucleotides with a frequency of 103–104 opposite all four template bases. Pol k is also a proficient extender of mispaired primer termini, an activity that may contribute to the rescuing of a stalled replication fork when mismatches fail to be removed by the exonuclease domain of replicative polymerases during normal DNA replication. Thus, whereas most DNA polymerases extend mispairs with about the same frequency as they misincorporate nucleotides, Pol k extends mispaired primer termini almost two orders of magnitude more efficiently than the frequency with which it misincorporates.

Does the structure offer any clues as to the ability of Pol k to extend mismatches better than other Y family polymerases? Extension of the primer terminus from a mismatch at the postinsertion T1-P1 position poses a different structural challenge than the insertion of a mismatch. The distorted DNA backbone geometry of a mismatch at T1-P1 will impact the position of the primer 30 OH in the active site, and thereby affect the nucleophilic attack on the incoming nucleotide. One could therefore envisage a unique set of residues in the Pol k active site that optimally aligns the primer 30 OH for the nucleophilic attack. However, Pol k residues in the vicinity of the putative 30 OH are similar to those in other Y family polymerases. As shown by Johnson and Beese in their crystallographic analysis of the high-fidelity Bacillus DNA polymerase I fragment (BF) bound to mispaired DNAs (Johnson and Beese, 2004), a mismatch at T1-P1 can also cause displacement of the template strand. In the BF structures, distortions in the template strand at T1 (and even as far away as T4) propagate to the preinsertion site and are predicted to interfere with the transfer of the unpaired template nucleotides to the insertion site. However, Pol k does not appear from our structure to differ from other Y family polymerases in its ability to propagate template-mediated distortion to the preinsertion site, as the DNA conformation in the vicinity of T1 is similar (B form) to that observed with other Y family polymerases. Also, the unpaired template nucleotides in the Pol k complex (at T1 and T2 positions) weave

shown in red, and the Mg2+ ion is shown as a blue sphere. On the right, the Mg2+ ion coordination geometry is shown in more detail along with the ligation distances (in A˚ units). Pink star marks the site of putative 30 OH.

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Figure 6. Modeling of DNA Lesions (A) Model of a cis-syn cyclobutane T-T dimer. A cis-syn T-T dimer (red) was modeled in the active sites of Pol k and Pol h with the 30 T at the templating position. Coordinates for the T-T dimer were obtained from the structure of Dpo4 complexed with a cys-syn T-T dimer (Ling et al., 2003). The yeast apo Pol h structure was superimposed onto that of the Pol k-DNA complex via the palm domain. The fingers domain and N-clasp are highlighted in yellow and dark blue, respectively, with residues apposed to the T-T dimer colored in green. The rest of the template and primer strands are shown in gray, and the incoming dNTP is omitted. In Pol k, the N-clasp and the fingers domain are in close proximity to the T-T dimer and the 50 T collides with F49 and M135. In contrast, Pol h can readily accommodate the 50 T of the T-T dimer because it lacks an equivalent N-clasp, and the fingers are positioned farther away from the templating strand. (B) Model of an HNE-dG adduct. An HNE-dG minor groove adduct (cyan) was modeled in the active sites of Pol k and Pol i at the postinsertion T1 position. The HNE-dG adduct is shown in the ring-open form with the active sites of Pol k and Pol i displayed as surface representations. Flanking DNA bases are omitted for clarity. Pol k is able to extend past the HNE-dG adduct at the T1 position, whereas the same lesion in Pol i blocks replication. From the model, the ring-open form of the HNE-dG adduct fits into the minor groove and is unobstructed by Pol k; the closest Pol k residues (F171, R175, and L197) are highlighted in white. In contrast, the minor groove is less accessible with Pol i and the long chain of the HNE-dG adduct clashes with residues Y39 and F125 on the Pol i molecular surface.

a convoluted path between the N-clasp and the fingers domain and appear, if anything, more resistant to transfer to the insertion site. Based on the apo Pol k structure, we had previously considered the possibility that the Pol k PAD is perhaps less constrained in its interactions with the template-primer, and thus a better ‘‘absorber’’ of DNA distortions at T1-P1 (Uljon et al., 2004). The Pol k ternary complex shows the opposite: a PAD held firmly on the template-primer because of its interactions with the N-clasp. Thus, among possible mechanisms, we favor the idea that Pol k extends mismatches proficiently because of its encirclement of the DNA that could in effect lock Pol k around the template-primer, as a means to keep it engaged on a sugar-phosphate backbone dis-

torted by a mismatch at T1-P1 and gaining time for the misaligned primer 30 OH to acquire proper alignment for the nucleophilic attack. Consistent with this idea, we show here that the N-clasp enhances the ability of Pol k to bind DNA and that it is crucial for its DNA polymerase activity. Pol k68–526 (lacking the N-clasp and the first 18 residues), for example, is severely impaired in its DNA synthesis activity from both matched and mismatched primer termini. Interestingly, Pol k1–526 is better (5- to 10-fold) in its ability to extend from mismatched primer termini than Pol k19–526, and Pol k1–526 also binds DNA somewhat tighter than Pol k19–526 (KD of 100 nM for Pol k1–526 versus a KD of 170 nM for Pol k19–526). Thus, although the Nclasp is apparently indispensable for DNA encirclement,

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the first 18 residues positively modulate the ability of the N-clasp for DNA binding and for mismatch extension. The proficient encirclement of DNA at the template-primer junction by the N-clasp aided by the first 18 residues could be instrumental in increasing the time ‘‘window’’ for the misaligned primer 30 OH to acquire the proper alignment for catalysis. The N-terminal 18 amino acids could potentially (based on the structure) interact with bases in the major groove and/or the sugar-phosphate backbone for added thermodynamic stability. From the structure, the inability of Pol k to insert nucleotides opposite the 30 T of a cis-syn T-T dimer stems from a constrained active-site cleft that cannot accommodate both Ts (connected by a covalent cyclobutane linkage) of the T-T dimer. This inability to insert opposite the 30 T of the T-T dimer appears to be confounded by the close proximity of the N-clasp to the active-site cleft, which overlaps with the 50 T of the dimer (Figure 6A). Although Pol k is unable to insert nucleotides opposite the 30 T of a T-T dimer, it can extend efficiently from a nucleotide inserted opposite the 30 T of the dimer by another polymerase: an activity that may contribute to the UV sensitivity of Pol k-deficient mouse cells (Ogi et al., 2002). As such, there is little steric interference when the 50 T of the T-T dimer is modeled at the templating position because the ‘‘preinsertion’’ nucleotide in this case can be kinked out of the active-site cleft. We show here that Pol k1–526 and Pol k19–526 are as effective as the full-length protein in extending from the 30 T of the cis-syn T-T dimer, but that Pol k68–526 lacking the N-clasp and the first 18 residues is severely impaired. The inability of Pol k68–526 to extend from the cis-syn T-T dimer likely derives from its poorer DNA binding (KD of 730 nM), and not because of any direct role for the N-clasp or the first 18 amino acids in stabilizing the 50 T of the T-T dimer at the templating position. The ability of Pol k to work in conjunction with another polymerase is best exemplified in the replication of the minor groove g-HOPdG adduct. The reaction of acrolein, an a,b-unsaturated aldehyde, with the N2 of dG followed by ring closure at N1 leads to the formation of the cyclic gHOPdG, which is a strong block to nucleotide incorporation by Pol k, but the polymerase can carry out efficient extension from a C nucleotide incorporated opposite gHOPdG by Pol i (Washington et al., 2004). From the structure, the inability of Pol k to insert a nucleotide opposite g-HOPdG likely stems from the disruption of the WatsonCrick edge of dG by the adduct and incompatibility with the Watson-Crick base pair observed in the active-site cleft. Pol i, on the other hand, can incorporate C opposite g-HOPdG because of its ability to rotate template purines from anti to syn conformation for productive Hoogsteen base-pairing (Nair et al., 2004, 2005a, 2006). Following the incorporation of C opposite cyclic g-HOPdG (in syn conformation), NMR studies have shown that the adduct is converted to a ring-open conformation that can partake in standard Watson-Crick base-pairing (de los Santos et al., 2001). Thus, following the translocation of the ringopen form of g-HOPdG to the postinsertion T1-P1 position,

Pol k can extend from it with the N2-3-hydroxypropyl chain projecting (unhindered) in the minor groove. In addition to acrolein, peroxidation of lipids in cells produces a variety of other reactive aldehydes, including trans-4-hydroxy-2-noenal (HNE) that conjugates with the N2 group of guanine (Chung et al., 1999; Esterbauer et al., 1991; Vaca et al., 1988). Because of its increased size, an HNE-dG adduct is potentially more blocking of replication than g-HOPdG. The ring-open form of HNE-dG (at T1 position) introduces a much more complex chain in the minor groove than g-HOPdG. Pol k can readily extend from the ring-open form of HNE-dG, but Pol i is strongly inhibited (Wolfle et al., 2006). The opposing abilities of Pol k and Pol i in extending HNE-dG appear to correlate to the fact that the minor groove edge of the postinsertion T1 nucleotide is less obstructed in the Pol k complex than in the Pol i complex (Figure 6B). In all, the sequential action of Pol i and Pol k could provide an important pathway for the efficient and error-free bypass of N2-dG adducts generated from endogenous cellular reactions such as lipid peroxidation. In conclusion, Y family polymerases have proven to be remarkably diverse in their functions and in strategies for replicating through DNA lesions. Pol i, for example, has emerged as specialized for Hoogsteen base-pairing, whereby templating A or G is driven to the syn conformation (Nair et al., 2004, 2005a, 2006). Rev1 adopts a radically different lesion-bypass strategy, where the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base (Nair et al., 2005b). Pol h is alone among eukaryotic polymerases in its proficient ability to replicate through UV-induced cissyn T-T dimers (Johnson et al., 1999b). Pol k, we show here, has a restrictive active-site cleft that seems to underlie both its better fidelity on undamaged DNAs and its inability to replicate through cis-syn T-T dimers. However, the most striking difference between Pol k and the other Y family polymerases is its near encirclement of DNA, which may underlie its ability to extend mismatches. Accordingly, Pol k becomes progressively less efficient in DNA binding and mismatch extension as the N-clasp and the first 18 residues are deleted. In addition, and importantly, the absence of any steric hindrance in the minor groove at the template-primer junction would enable Pol k to extend from nucleotides incorporated opposite various minor groove DNA lesions by another polymerase. EXPERIMENTAL PROCEDURES Pol k Truncations and Protein Purification Pol k1–526, Pol k19–526, and Pol k68–526 constructs were made as previously described (Uljon et al., 2004). The Pol k37–526 and Pol k47–526 constructs were generated by PCR amplification of the Pol k19–526 construct using primers to amplify regions corresponding to the residues indicated. Each construct was confirmed by sequence analysis and expressed as a GST fusion protein: Pol k1–526, Pol k19–526, and Pol k68–526 were expressed in yeast strain BJ5464, while the Pol k37–526 and Pol k47–526 fusion proteins were expressed in E. coli BL21Gold codon+ cells (Stratagene). The proteins were purified in

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a similar manner to that described previously for the Pol k68–526 construct (Uljon et al., 2004), except for the inclusion of an extra ion-exchange chromatography step before gel filtration. Selenomethionine (SeMet)-labeled Pol k19–526 was expressed in E. coli B834 methionine auxotrophic cells grown in M9 minimal medium supplemented with SeMet instead of methionine, and then purified in a manner similar to that used for the native protein. DNA Synthesis Assays and Steady-State Kinetic Analyses For primer extension assays with full-length or truncated Pol k proteins, we used 45 nucleotide primers (50 -GTTTTCCCAGTCACGAC GATGCTCCGGTACTCCAGTGTAGGCATN-30 , where N denotes either a G, A, T, or C) labeled with 32P at the 50 end and annealed to a DNA template. For extension from the above primers paired with a G residue, the 52-mer template oligonucleotide 30 -CAAAAGGGTCAG TGCTGCTACGAGGCCATGAGGTCACATCCGTAGTATGCTT-50 was used. To examine extension from an A or a G paired with the 30 T of a T-T dimer, or from an equivalent nondamaged T residue, the 75mer template 30 -CAAAAGGGTCAGTGCTGCTACGAGGCCATGAGGT CACATCCGTATTATGCTTGAGAATCTGTAACCACTGAACGA-50 was used, in which the underlined Ts indicate the position of the T-T dimer. The standard polymerase reaction was used and contained 25 mM each of dATP, dGTP, dTTP, and dCTP, and either 0.5 nM protein (Pol k1–870, Pol k1–526, or Pol k19–526) or 50 nM protein (Pol k68–526). Reactions were carried out for 5 min at 37 C. For steady-state kinetic experiments, only dATP, the next correct nucleotide, was added at concentrations ranging from 0.025–500 mM and reactions contained 0.05 nM protein. Reactions were carried out for 2.5 or 5 min at 37 C, and steady-state kinetic parameters were determined as described (Johnson et al., 2006). Cocrystallization A 13 nt primer was synthesized with a dideoxycytosine at its 30 end (50 GGGGGAAGGACCddC-30 ) and annealed with an 18 nt template (50 TTCCAGGGTCCTTCCCCC-30 ) to yield a 13 nt/18 nt primer/template. Both oligonucleotides were first purified by reverse-phase HPLC. Pol k19–526 (0.5 mM final concentration) was incubated with a 2-fold excess of the 13 nt/18 nt primer/template in a buffer containing 25 mM HEPES (pH 7.0), 200 mM NaCl, 10 mM dTTP, 10 mM MgCl2, and 1 mM TCEP (Tris [2-carboxyethyl] phosphine hydrochloride). Cocrystals were obtained from solutions containing 12% PEG 5000 monomethyl ether (w/v) (MME), 200 mM ammonium or potassium acetate, and 100 mM NaCl and were buffered at pH 6.5 or 6.2. The cocrystals belong to space group C2221 with cell dimensions a = 116.15 A˚, b = 152.47 A˚, c = 217.0 A˚, a = 90 , b = 90 , and g = 90 . Crystals of SeMet Pol k19–526 ternary complex were obtained under similar conditions as the native, but they tended to be smaller in size. For data collection, native and SeMet cocrystals were cryoprotected by 5 min soaks in mother liquor solutions containing 5%–30% PEG 350 MME, and then flash frozen in liquid nitrogen. Structure Determination and Refinement X-ray data on cryocooled native and SeMet cocrystals were measured at the APS on beamline 17-ID. An initial native data set (Native 1) to 3.2 A˚ was indexed and integrated using DENZO and reduced using SCALEPACK (Otwinowski and Minor, 1997). These data were used to obtain an MR solution using the apo Pol k structure (without the PAD) as a search model. Specifically, the program PHASER (McCoy et al., 2005) gave a unique MR solution, containing two complexes in the asymmetric unit, which could be rigid body refined to an Rfree of 47% using CNS (Brunger et al., 1998). To validate the MR solution, low-resolution data were measured on an SeMet crystal (5.5 A˚, at l = 0.979A˚) and used to compute an anomalous difference Fourier map, which showed the seleniums at the expected positions. Following additional purification of the polymerase by gel filtration and refinement of the crystallization conditions (most notably, a change in salt from ammonium acetate to potassium acetate and a change in pH

from 6.5 to 6.2), a second native data set (Native 2) extending to 3.05 A˚ was collected, which was more complete and merged with better statistics than Native 1 (Table 2). Interestingly, these Native 2 data were not isomorphous with Native 1 data and led to a different MR solution, using the program AMoRe (Navaza, 1994). Rigid body refinement with CNS gave an Rfree of 44%, and the electron density maps were better quality than the Native 1 maps, showing, for example, the clear presence of the incoming dTTP. The Rfree for the starting model dropped to 39% through iterative rounds of model building in O (Jones et al., 1991) followed by energy minimization and rigid body refinement in CNS. Several cycles of simulated annealing, positional and B factor refinements with CNS, and model building in O lowered the Rfree to 29.4%, with an Rcryst of 23.1%. The final Pol k ternary complex model includes residues 33–224 and 282–517 for protein molecule A, and residues 21–224 and 282–517 for molecule B; DNA nucleotides 3–15 and 4–13 for template (T) and primer (P) strands bound to protein A, and nucleotides 3–17 and 2–13 for template (T) and primer (P) strands bound to protein B; two incoming dTTP nucleotides; and 21 water molecules. Approximately 11% and 15% of the residues are built as alanines in protein molecules A and B (primarily at the N terminus), respectively, because of their undefined side-chain densities. Structural Analysis The model has good stereochemistry, as shown by PROCHECK (Laskowski et al., 1993), with 80%–85% of the residues in the most favored regions of the Ramachandran plot and no residues in the disallowed regions (Table 1). Secondary structure assignments were conducted using PROCHECK. The buried surface areas were calculated using CNS with a probe radius of 1.4 A˚. Figures were prepared using PyMol (Delano, 2002). KD Determination by Fluorescence Anisotropy 6-carboxyfluorescein (6-FAM)-labeled template (50 -TTCCAGGGTC CTTCCCCC-30 ) and primer (50 -GGGGGAAGGACCC-30 ) were purchased PAGE purified from IDT Technologies (Coralville, IA). Duplexes were formed by heating to 95 C a mixture of one equivalent of the 6-FAM-labeled strand with one equivalent of the complementary strand and permitting the sample to cool to room temperature. Fluorescence emission intensities were collected on a Panvera Beacon 2000 fluorescence polarization system (at 25 C). 6-FAMlabeled oligonucleotides were excited at 490 nm, and the resulting emission was passed through a 520 nm cutoff filter. Readings were taken at all four combinations of vertical (v) and horizontal (h) polarizer settings using the L format, taken over a 10 s integration time, and averaged. Anisotropy values were calculated from the equation A = ðIvv  Ivh Þ=ðIvv + 2Ivh Þ where Ivv and Ivh are the recorded intensities of the vertical and horizontal polarized light. 6-FAM-labeled oligonucleotides (5 nM) were added to increasing concentrations of the Pol k deletions (1–526, 19–526, 37–526, 47– 526, and 68–526 [0.457 nM to 15 mM]), and the samples were left to equilibrate at room temperature for 30 min before fluorescence anisotropy values were measured. Anisotropy values were referenced against a blank sample of buffer (25 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 1 mM DTT, and 10% glycerol) at the beginning of each experiment to account for background correction. Measurements were taken three times at each concentration, and the anisotropy values were averaged. Anisotropy values were normalized by first subtracting the anisotropy value with no protein added and then dividing by the maximum anisotropy value for a particular enzyme series. Anisotropy values were then plotted versus enzyme concentration, and the data were fitted by nonlinear least squares regression, using Origin 7 (OriginLab), to the following quadratic equation:

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q = ½ðKD + Do + Eo Þ  fðKD + Do + Eo Þ2  4Do Eo g1=2 =2Do

Molecular Cell Structure of DNA Polymerase k Ternary Complex

where q is the fraction of DNA duplex bound, Do is the total concentration of DNA duplex, Eo is the total enzyme concentration, and KD is the dissociation constant.

Supplemental Data Supplemental Data include one figure and can be found with this article online at http://www.molecule.org/cgi/content/full/25/4/601/DC1/.

ACKNOWLEDGMENTS We thank the staff at APS (beamline 17-ID) and BNL (beamlines X12C and X6A) for facilitating X-ray data collection. We thank T. Edwards for help with crystallography. This work was supported by grant CA094006 from the NIH (A.K.A. and L.P.). Received: July 5, 2006 Revised: October 22, 2006 Accepted: January 17, 2007 Published: February 22, 2007

Johnson, R.E., Washington, M.T., Haracska, L., Prakash, S., and Prakash, L. (2000b). Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature 406, 1015–1019. Johnson, R.E., Washington, M.T., Prakash, S., and Prakash, L. (2000c). Fidelity of human DNA polymerase eta. J. Biol. Chem. 275, 7447–7450. Johnson, R.E., Trincao, J., Aggarwal, A.K., Prakash, S., and Prakash, L. (2003). Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases. Mol. Cell. Biol. 23, 3008–3012. Johnson, R.E., Prakash, L., and Prakash, S. (2006). Yeast and human translesion DNA synthesis polymerases: expression, purification, and biochemical characterization. Methods Enzymol. 408, 390–407. Jones, T.A., Zou, J.-Y., and Cowan, S.W. (1991). Improved methods for building models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. Kim, S.R., Maenhaut-Michel, G., Yamada, M., Yamamoto, Y., Matsui, K., Sofuni, T., Nohmi, T., and Ohmori, H. (1997). Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc. Natl. Acad. Sci. USA 94, 13792–13797.

REFERENCES Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R., Jiang, W., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. Chung, F.L., Zhang, L., Ocando, J.E., and Nath, R.G. (1999). Role of 1,N2-propanodeoxyguanosine adducts as endogenous DNA lesions in rodents and humans. IARC Sci. Publ. 150, 45–54. Delano, W.L. (2002). The PyMol Molecular Graphics System (San Carlos, CA: Delano Scientific LLC). de los Santos, C., Zaliznyak, T., and Johnson, F. (2001). NMR characterization of a DNA duplex containing the major acrolein-derived deoxyguanosine adduct gamma-OH-1,-N2-propano-20 -deoxyguanosine. J. Biol. Chem. 276, 9077–9082. Published online October 27, 2000. 10.1074/jbc.M009028200. DeLucia, A.M., Grindley, N.D., and Joyce, C.M. (2003). An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a ‘steric gate’ residue for discrimination against ribonucleotides. Nucleic Acids Res. 31, 4129–4137. Doublie, S., Tabor, S., Long, A.M., Richardson, C.C., and Ellenberger, T. (1998). Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A˚ resolution. Nature 391, 251–258. Esterbauer, H., Schaur, R.J., and Zollner, H. (1991). Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81–128. Haracska, L., Johnson, R.E., Unk, I., Phillips, B.B., Hurwitz, J., Prakash, L., and Prakash, S. (2001). Targeting of human DNA polymerase iota to the replication machinery via interaction with PCNA. Proc. Natl. Acad. Sci. USA 98, 14256–14261. Johnson, S.J., and Beese, L.S. (2004). Structures of mismatch replication errors observed in a DNA polymerase. Cell 116, 803–816. Johnson, R.E., Kondratick, C.M., Prakash, S., and Prakash, L. (1999a). hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285, 263–265. Johnson, R.E., Prakash, S., and Prakash, L. (1999b). Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta. Science 283, 1001–1004. Johnson, R.E., Prakash, S., and Prakash, L. (2000a). The human DINB1 gene encodes the DNA polymerase Poltheta. Proc. Natl. Acad. Sci. USA 97, 3838–3843.

Kobayashi, S., Valentine, M.R., Pham, P., O’Donnell, M., and Goodman, M.F. (2002). Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment. J. Biol. Chem. 277, 34198–34207. Kokoska, R.J., Bebenek, K., Boudsocq, F., Woodgate, R., and Kunkel, T.A. (2002). Low fidelity DNA synthesis by a y family DNA polymerase due to misalignment in the active site. J. Biol. Chem. 277, 19633– 19638. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. A47, 110–119. Li, Y., Korolev, S., and Waksman, G. (1998). Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 17, 7514–7525. Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001). Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102. Ling, H., Boudsocq, F., Plosky, B.S., Woodgate, R., and Yang, W. (2003). Replication of a cis-syn thymine dimer at atomic resolution. Nature 424, 1083–1087. Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K., and Hanaoka, F. (1999). The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399, 700–704. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C., and Read, R.J. (2005). Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464. Nair, D.T., Johnson, R.E., Prakash, S., Prakash, L., and Aggarwal, A.K. (2004). Replication by human DNA polymerase-iota occurs by Hoogsteen base-pairing. Nature 430, 377–380. Nair, D.T., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K. (2005a). Human DNA polymerase iota incorporates dCTP opposite template G via a G.C+ Hoogsteen base pair. Structure 13, 1569–1577. Nair, D.T., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K. (2005b). Rev1 employs a novel mechanism of DNA synthesis using a protein template. Science 309, 2219–2222. Nair, D.T., Johnson, R.E., Prakash, L., Prakash, S., and Aggarwal, A.K. (2006). An incoming nucleotide imposes an anti to syn conformational change on the templating purine in human DNA polymerase-i active site. Structure 14, 749–755.

Molecular Cell 25, 601–614, February 23, 2007 ª2007 Elsevier Inc. 613

Molecular Cell Structure of DNA Polymerase k Ternary Complex

Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallogr. A50, 157–163. Ogi, T., Kato, T., Jr., Kato, T., and Ohmori, H. (1999). Mutation enhancement by DINB1, a mammalian homologue of the Escherichia coli mutagenesis protein dinB. Genes Cells 4, 607–618. Ogi, T., Shinkai, Y., Tanaka, K., and Ohmori, H. (2002). Polkappa protects mammalian cells against the lethal and mutagenic effects of benzo[a]pyrene. Proc. Natl. Acad. Sci. USA 99, 15548–15553. Ohashi, E., Ogi, T., Kusumoto, R., Iwai, S., Masutani, C., Hanaoka, F., and Ohmori, H. (2000). Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa. Genes Dev. 14, 1589–1594. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Prakash, S., Johnson, R.E., and Prakash, L. (2005). Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74, 317–353. Silvian, L.F., Toth, E.A., Pham, P., Goodman, M.F., and Ellenberger, T. (2001). Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus. Nat. Struct. Biol. 8, 984–989.

Vaca, C.E., Wilhelm, J., and Harms-Ringdahl, M. (1988). Interaction of lipid peroxidation products with DNA. A review. Mutat. Res. 195, 137– 149. Washington, M.T., Johnson, R.E., Prakash, S., and Prakash, L. (2000). Accuracy of thymine-thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase eta. Proc. Natl. Acad. Sci. USA 97, 3094–3099. Washington, M.T., Johnson, R.E., Prakash, L., and Prakash, S. (2002). Human DINB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini. Proc. Natl. Acad. Sci. USA 99, 1910–1914. Washington, M.T., Prakash, L., and Prakash, S. (2003). Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase eta. Proc. Natl. Acad. Sci. USA 100, 12093–12098. Washington, M.T., Minko, I.G., Johnson, R.E., Wolfle, W.T., Harris, T.M., Lloyd, R.S., Prakash, S., and Prakash, L. (2004). Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa. Mol. Cell. Biol. 24, 5687–5693.

Steitz, T.A. (1999). DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398.

Wolfle, W.T., Washington, M.T., Prakash, L., and Prakash, S. (2003). Human DNA polymerase kappa uses template-primer misalignment as a novel means for extending mispaired termini and for generating single-base deletions. Genes Dev. 17, 2191–2199.

Trincao, J., Johnson, R.E., Escalante, C.R., Prakash, S., Prakash, L., and Aggarwal, A.K. (2001). Structure of the catalytic core of S. cerevisiae DNA polymerase eta: implications for translesion DNA synthesis. Mol. Cell 8, 417–426.

Wolfle, W.T., Johnson, R.E., Minko, I.G., Lloyd, R.S., Prakash, S., and Prakash, L. (2006). Replication past a trans-4-hydroxynonenal minorgroove adduct by the sequential action of human DNA polymerases iota and kappa. Mol. Cell. Biol. 26, 381–386.

Trincao, J., Johnson, R.E., Wolfle, W.T., Escalante, C.R., Prakash, S., Prakash, L., and Aggarwal, A.K. (2004). Dpo4 is hindered in extending a G.T mismatch by a reverse wobble. Nat. Struct. Mol. Biol. 11, 457– 462.

Zhou, B.L., Pata, J.D., and Steitz, T.A. (2001). Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain. Mol. Cell 8, 427–437.

Uljon, S.N., Johnson, R.E., Edwards, T.A., Prakash, S., Prakash, L., and Aggarwal, A.K. (2004). Crystal structure of the catalytic core of human DNA polymerase kappa. Structure 12, 1395–1404.

Accession Numbers The coordinates have been deposited to the RCSB Protein Data Bank with accession code 2OH2.

614 Molecular Cell 25, 601–614, February 23, 2007 ª2007 Elsevier Inc.