Under optimal conditions, the fidelity of DNA replication is extremely high. Indeed, it is estimated that, on average, only one error occurs in every 1010 bases replicated. However, as living organisms are continually subjected to a variety of endogenous and exogenous DNA-damaging agents, optimal conditions rarely prevail in vivo. Although all organisms have evolved elaborate repair pathways to deal with such damage, the repair pathways rarely operate with 100 percent efficiency. As a consequence, the persisting DNA lesions are replicated, but with much lower fidelity than undamaged DNA. The general aim of this project is therefore to understand the molecular mechanisms by which mutations are introduced into damaged DNA. The process is commonly referred to as translesion DNA synthesis (TLS) or translesion replication (TR) and is thought to be facilitated by one or more of the Y-family of DNA polymerases that are phylogenetically conserved from bacteria to humans. As in past years, we have continued to investigate TLS in all three kingdoms of life: bacteria, archaea, and eukaryotes.
Translesion replication in prokaryotes
Dotter, Gupta, Karata, Mead, Woodgate; in collaboration with Goodman
In E. coli, efficient translesion replication of many DNA lesions occurs only when the UmuC protein physically interacts with a dimer of UmuD´ to form a heterotrimeric complex of UmuD´2C (known as E. coli pol V). Because pol V is a low-fidelity enzyme, its activities within the cell are strictly controlled at several levels. For example, the umu genes are arranged in an operon and are negatively regulated at the transcriptional level by the SOS repressor LexA. Transcriptional regulation is, however, insufficient, and the cellular levels of UmuD and UmuC are kept to a minimum through their rapid Lon-mediated proteolytic degradation. After cellular DNA damage, RecA protein nucleates on regions of single-stranded DNA and mediates the post-translational self-cleavage of LexA, leading to its inactivation and the derepression of genes in the LexA-regulon, including umuD and umuC. Interestingly, UmuD undergoes a mechanistically similar self-cleavage reaction. In the case of UmuD, cleavage of its N-terminal 24 amino acids converts it to UmuD´ and activates it for its role in SOS mutagenesis. UmuD and UmuD´ both form homodimers but, when mixed together, preferentially associate as a UmuD/D´ heterodimer, with UmuD´ susceptible to proteolysis by the ClpXP serine protease. Degradation of the mutagenically active UmuD´ subunit therefore helps return cells to a resting state once cellular DNA damage has been repaired and the need for pol V has abated.
In vitro replication assays reveal that regulation of the catalytic activity of pol V is also modulated through several protein-protein interactions. For example, pol V does not catalyze TLS alone but is instead an essential component of a multiprotein “mutasome” complex composed of RecA protein, beta sliding-clamp, and SSB. In collaboration with Myron Goodman, we have investigated in detail the nature of the interactions between RecA and pol V. We found that pol V and RecA interact physically through two distinct mechanisms. The first occurs when pol V binds to RecA through its UmuC subunit in the absence of DNA and ATP while the second occurs through pol V’s UmuD´ subunit in the presence of DNA and ATP. Pol V–catalyzed synthesis with mutant RecAs with an increased affinity for ssDNA revealed that any additional RecA bound to DNA inhibits normal DNA synthesis and TLS, suggesting that a RecA nucleoprotein filament is not likely to be a prerequisite for SOS mutagenesis. Pol V failed to synthesize DNA with a RecA mutant (RecA1730) that is defective in promoting SOS mutagenesis in vivo, suggesting that RecA may serve as an obligate accessory factor for stimulating pol V activity in vitro and in vivo.
Although the principal biological role of pol V appears to involve TLS and the generation of mutational diversity to cope with stress, we recently discovered that pol V possesses intrinsic apurinic/apyrimidinc 5´-deoxyribose phosphate (AP/5´-dRP) lyase activity. We also identified a similar activity in the distantly related E. coli Y-family polymerase pol IV. Both polymerases catalyze cleavage of the phosphodiester backbone at the 3´-side of an apurinic/apyrimidinic (AP) site as well as removal of a 5´-deoxyribose phosphate (dRP) at a pre-incised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase zeta, which is a key enzyme in short-patch base excision repair. Pol IV forms a covalent Schiff’s base intermediate with substrate DNA that is trapped by sodium borohydride. In contrast, we did not observe an NaBH4-trapped intermediate for pol V even though the lyase-specific activity of pol V is slightly higher than that of pol IV. Incubation of pol V (UmuD´2C) with a molar excess of UmuD drives an exchange of subunits to form UmuD´/D and insoluble UmuC, causing inactivation of polymerase and lyase activities. The concomitant loss of both activities is strong evidence that pol V contains a bona fide lyase activity.
Schlacher K, Leslie K, Wyman C, Woodgate R, Cox MM, Goodman MF. DNA polymerase V and RecA, a minimal mutasome. Mol Cell 2005;17:561-572.
Shen X, Woodgate R, Goodman MF. Lyase activities intrinsic to Escherichia coli polymerases IV and V. DNA Repair 2005;4:1368-1373.
Structural analysis of lesion bypass
Vaisman, Plosky, Woodgate; in collaboration with Kunkel, Yang
We previously identified and cloned a Y-family DNA polymerase from the thermostable archaeon Sulfolobus solfataricus called DNA polymerase IV (Dpo4). In a collaborative study with Wei Yang, we used x-ray diffraction to solve the crystal structure of Dpo4 in a ternary complex with an undamaged DNA template DNA and an incoming dexoyribose nucleoside triphosphate. The investigation revealed that, like all DNA polymerases, Dpo4 possesses a topology similar to a right hand with protein domains that resemble “fingers,” a “palm,” and a “thumb.” However, the domains are all stubby, and the active site of the enzyme is large and solvent-exposed. Dpo4 also possesses a unique domain called the “little finger” that helps the enzyme bind to DNA. To understand how lesion bypass occurs at the molecular level, we crystallized an abasic site in the active site of Dpo4. Even though the lesion is small, it nevertheless blocks replication by cellular replicases, as the base is physically absent and there is no genetic information to instruct continued DNA synthesis. We determined the crystal structures of Dpo4 complexed with five abasic site–containing DNA substrates and discovered that translesion synthesis is template-directed, with the abasic site looping out and the incoming nucleotide opposite the base 5´ to the lesion. The ensuing DNA synthesis generates a -1 frameshift when the abasic site remains extra-helical. We also observed template realignment during primer extension, which resulted in base substitutions or even +1 frameshifts. In the case of a +1 frameshift, the extra nucleotide was accommodated in the solvent-exposed minor groove.
In addition, we crystallized the structure of an unproductive Dpo4 ternary complex, whose structure suggested that the flexible little finger (LF) domain facilitates DNA orientation and translocation during translesion synthesis. We investigated such a possibility further by making chimeras of Dpo4 and Dbh in which their LF domains were interchanged. Dpo4 and Dbh originate from two closely related strains of Sulfolobaceae, yet the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, but Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base-pair substitutions while Dbh predominantly makes single-base deletions. Interestingly, replacement of the LF domain of Dbh with that of Dpo4 makes the enzymatic properties of the chimeric enzyme more Dpo4-like in that the enzyme is more processive and can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer (CPD). In addition, the chimera predominantly makes base-pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4LFDbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies therefore indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each Y-family member.
We also recently solved the crystal structures of Dpo4 complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. The structures suggest two mechanisms by which Dpo4 may reject an incorrect incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and, as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may therefore be a key step in catalysis. Second, we hypothesize that the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, we demonstrated that Dpo4 exhibits robust pyrophosphorolysis activity and is able to degrade a primer strand in the presence of an incorrect incoming nucleotide.
Boudsocq F, Kokoska RJ, Plosky BS, Vaisman A, Ling H, Kunkel TA, Yang W, Woodgate R. Investigating the role of the little finger domain of Y-family DNA polymerases in low-fidelity synthesis and translesion replication. J Biol Chem 2004;279:32932-32940.
Ling H, Boudsocq F, Woodgate R, Yang W. Snapshots of replication through an abasic lesion; structural basis for base substitutions and frameshifts. Mol Cell 2004;13:751-762.
Vaisman A, Ling H, Woodgate R, Yang W. Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO J 2005;24:2957-2967.
Identification and characterization of novel Dop4-like enzymes in archaea
McDonald, Woodgate; in collaboration Ballantyne, Cadet
The ability to detect DNA polymorphisms with molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role in the criminal justice system, but one of the technology’s limiting factors is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. While Dpo4 can perform PCR at moderately high temperatures, it is not as thermostable or processive as Taq polymerase. We were therefore interested in identifying novel Dpo4-like enzymes that might possess greater processivity or thermostability than Dpo4. To do so, we used the amino acid sequences of S. acidocaldarius Dbh (Sa) and S. solfataricus Dpo4 (Sso) as well as a hypothetical Dpo4-like protein encoded within the S. tokodaii genome to design degenerate PCR primers that we used to PCR-amplify additional Dpo4-like genes from strains of Sulfolobaceae that grow at temperatures ranging from 75 to 90°C. We were successful in identifying five new pol IV homologues from Acidianus infernus (Ai), Sulfolobus shibatae (Ssh), Sulfolobus tengchongensis (Ste), Stygiolobus azoricus (Saz), and Sulfurisphaera ohwakuensis(Soh). To characterize the biochemical properties of the new pol IV–like enzymes, we overexpressed them in E. coli and purified them by using a scheme similar to but somewhat simplified from that previously reported for Dpo4. Such an approach allowed us to purify between 1 and 5 mg of each polymerase readily from just 1 to 2L of E. coli cell culture. Once we had purified the polymerases to greater than 95 percent homogeneity, we characterized their relative processivity and lesion-bypassing activities. Of the five novel enzymes, Ste Dpo4 was the most robust and appeared to be as good as, if not better, than Dpo4 at high-temperature processive DNA synthesis. The Ai-Dpo4 enzyme was not as processive, but, by interchanging its “little finger” domain with that of Sso-Dpo4 or Ste-Dpo4, we increased its processivity considerably; the Ai/Sso and Ai/Ste Dpo4-like chimeras were the most efficient of the novel enzymes at bypassing a variety of DNA lesions.
To test the usefulness of the novel enzymes, we irradiated human genomic DNA with UVC light and attempted to amplify Alu short interspersed elements. Taq alone gave an Alu signal that was barely detectable. Remarkably, we obtained a 40-fold increase in the amplicon when we used a blend of Taq with Sso Dpo4 in the PCR reaction. Our findings therefore have immediate and obvious applications in forensic science and the analysis of ancient DNA samples.
Translesion replication in Saccharomyces cerevisiae
McDonald, Woodgate; in collaboration Lawrence
In addition to a variety of mechanisms that repair damage to its genome, the yeast Saccharomyces cerevisiae possesses mechanisms that lead to the tolerance of DNA damage by promoting translesion replication. Genetic experiments have previously implicated DNA polymerases eta, encoded by RAD30, and zeta, encoded by REV3 and REV7, as required for the bypass of lesions that would otherwise cause the replicase to stall. In addition to these enzymes, translesion replication often requires the Rev1 protein, which appears to be essential for pol zeta activity, and Pol32p, a subunit of DNA polymerase delta required for induced mutagenesis. The various functions of these proteins in translesion replication remains unclear. Therefore, we investigated their roles in the in vivo bypass of an abasic site, 6-4 photoadduct (6-4PP) and cis-syn cyclobutane dimer (CPD) by transforming isogenic yeast strains deleted for RAD30, REV3, REV1, or POL32 with duplex plasmids carrying one of these DNA lesions within a 28-nucleotide single-stranded region. Bypass frequencies and nucleotide insertion spectra from our experiments showed that pol eta is only rarely involved in the bypass of the abasic site or the 6-4PP but was, as expected, solely responsible for the bypass of the CPD. The insertion of dG opposite the 3´ T of the 6-4PP, characteristic of Pol eta, significantly declined from 10 percent of all insertions in the wild-type to 4 percent in the rad30 deletion strain, showing some involvement of Pol eta in such events. However, the result also suggests that another enzyme can generate the mutations, and we hypothesize that pol zeta is responsible for insertion in all other bypass events. Pol delta is also clearly involved in mutagenesis, given that strains lacking Pol32 are known to be deficient in mutagenesis and, in keeping with this observation, a pol32 deletion strain shows as little bypass of the 6-4PP photoadduct or the abasic sites as those strains eficient in pol zeta or Rev1. Thus, TLS in Saccharomyces cerevisiae is complex and appears often to require a combination of one or more polymerases to facilitate the bypass of many DNA adducts.
Gibbs PEM, McDonald JP, Woodgate R, Lawrence CW. The relative roles in vivo of Saccharomyces cerevisiae Pol eta, Pol zeta, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct, and T-T cis-syn cyclobutane dimer. Genetics 2005;169:575-582.
Characterization of human DNA polymerases iota and eta
Chumakov, McDonald, McLenigan, Mead, Plosky, Vaisman, Woodgate; in collaboration Gearhart, West
Humans possess four Y-family polymerases; pol eta, pol iota, pol kappa, and Rev1. Given that we identified the enzyme, pol iota is of particular interest to us. Pol iota is a paralogue of pol eta, but unlike pol eta, which bypasses UV-induced CPDs efficiently and accurately, pol iota demonstrates a somewhat varied ability to bypass CPDs, with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. While defects in pol eta lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant (XP-V) phenotype, the biological function of pol iota remains to be determined.
Our recent studies reveal that human pol iota and the proliferating cell nuclear antigen (PCNA) physically interact and that such interaction stimulates the processivity of pol iota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP-box) of pol iota or the interdomain connector loop of PCNA diminished the binding between pol iota and PCNA and concomitantly reduced PCNA-dependent stimulation of pol iota activity. Furthermore, the pol iota-PIP box mutant failed to accumulate into replication foci after cellular DNA damage, indicating that an interaction between pol iota and PCNA is essential for foci formation.
Owing to their “cavernous” active sites that can accommodate a wide variety of geometric distortions, Y-family DNA polymerases are considerably more error-prone than high-fidelity replicases. Therefore, it is not surprising that the in vivo activity of these polymerases is tightly regulated so as to minimize their inadvertent access to primer termini. One such mechanism that we discovered in the past year relies on a direct interaction between human pol iota and pol eta with ubiquitin. In particular, we identified a region within each polymerase that appears essential for polymerase-ubiquitin interactions, including binding to ubiquitinated-PCNA and free polyubiquitin chains. Polymerase mutants that are unable to interact with ubiquitin highlight the biological importance of the polymerase-ubiquitin interaction, given that they exhibit significantly lower levels of replication foci in response to DNA damage. Thus, pol eta’s and pol iota’s ability to bind to ubiquitin is a key step in delivering the TLS polymerases to sites of DNA damage, where they can facilitate lesion bypass.
Lesions in DNA pose a serious threat to the integrity of the genome as they stall replication forks. To overcome the catastrophic consequences associated with fork demise, TLS polymerases such as pol eta promote DNA synthesis past DNA lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. In collaboration with Stephen West, we used fractionated cell-free extracts and purified recombinant proteins to demonstrate that pol eta binds and extends DNA synthesis from D-loop recombination intermediates in which an invading strand serves as the primer for DNA extension. Extracts prepared from human XP-V cells defective for pol eta exhibited severely reduced D-loop extension activity. The ability of pol eta to promote D-loop extension is unusual; neither the replicative DNA polymerase delta nor pol iota could promote such a reaction. We also found that pol eta interacts with the RAD51 recombinase and that pol eta–mediated D-loop extension activity is stimulated by the presence of RAD51. Our results therefore indicate a novel dual function for pol eta at stalled replication forks: in the promotion of translesion synthesis and in the reinitiation of DNA synthesis by homologous recombination repair.
In addition to facilitating TLS and/or DNA synthesis during recombination, pol eta has been implicated in the generation of mutations in immunoglobulin genes. The current model postulates that the activation-induced cytidine deaminase (AID) deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G base pairs in immunoglobulin genes. The mechanism that generates mutations at A:T base pairs is, however, unclear, although such mechanism appears to require the MSH2-MSH6 mismatch repair heterodimer and DNA polymerase eta, as mutations of A:T are decreased in mice and humans lacking these proteins. In collaboration with Patricia Gearhart, we demonstrated that MSH2/6 and pol eta interact both physically and functionally. For example, MSH2 binds to pol eta in solution, and endogenous MSH2 associates with pol eta in cell extracts. Importantly, MSH2-MSH6 stimulated the catalytic activity of pol eta in vitro. Our observations suggest that the interaction between MSH2-MSH6 and DNA pol eta stimulates low-fidelity synthesis and the generation of mutations at bases located downstream of the initial dU lesion, including A:T pairs.
McIlwraith MJ, Vaisman A, Liu Y, Fanning E, Woodgate R, West SC. Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. Mol Cell 2005;20:783-792.
Vidal AE, Kannouche P, Podust VN, Yang W, Lehmann AR, Woodgate R. Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota. J Biol Chem 2004;279:48360-48368.
Wilson TM, Vaisman A, Martomo SA, Sullivan P, Lan L, Hanaoka F, Yasui A, Woodgate R, Gearhart PJ. MSH2-MSH6 stimulates DNA polymerase eta, suggesting a role for A:T mutations in immunoglobulin genes. J Exp Med 2005;201:637-645.
Role of DNA polymerase iota in humans with the xeroderma pigmentosum variant (XP-V) syndrome
Vaisman, Woodgate; in collaboration Iwai, Maher
Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only about 25 percent of mutations occur at thymine (T) while 75 percent are targeted to cytosine (C). The mutational spectrum changes dramatically in XP-V cells, which are devoid of pol eta, in which about 45 percent of mutations occur at Ts and about 55 percent at Cs. At present, it is unclear whether the C®T mutations represent true misincorporations opposite C or instead occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that has undergone deamination to uracil (U). To assess the possible role of human pol iota in the replicative bypass of such UV-photoproducts, we analyzed the efficiency and fidelity of pol iota–dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota–dependent bypass of a T-U CPD occurred more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3´ U of the T-U CPD only half as frequently as the correct Watson-Crick base, A. While pol iota generally extended the G:3´ U–CPD mispairs less efficiently than the correctly paired primer, pol iota–dependent extension was equal to or greater than that observed with human pol eta and pol kappa and S. cerevisiae pol zeta under the same assay conditions.
We believe that the unique pattern of misincorporations previously observed during the pol iota–dependent bypass of a T-T CPD and T-U CPD (described above) readily explains the abnormal spectrum of mutations observed in XP-V cells. For example, pol iota misincorporates G and T opposite the 3´ T of the T-T CPD, which would lead to the increase in the observed T®C and T®A substitutions. Similarly, pol iota misincorporates T opposite the 3´ U of the T-U CPD, which would give rise to the C®A mutations observed in XP-V cells. The decrease in C®T transitions can also be explained by the propensity of pol iota to “misincorporate” G opposite U, given that the “misincorporation” would be error-free if the U arose by deamination of C.
In collaboration with Veronica Maher, we set out to test the misincorporation hypothesis in vivo by transfecting an XP-V cell line with anti-sense to POLI and identifying two stable cell lines in which pol iota expression was reduced by about 50 percent. As hypothesized, the mutation frequency also decreased by about 50 percent in the anti-sense–expressing strains. Our results therefore indicate that, in XP-V cells, pol iota does indeed cause the high frequency of mutations and abnormal spectrum induced by UV-light.
Vaisman A, Takasawa K, Iwai S, Woodgate R. DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers. DNA Repair 2005;5:210-218.
Collaborators
Jack Ballantyne, PhD, University of Central Florida, Orlando, FL
Jean Cadet, PhD, CEA-Grenoble, Grenoble, France
Pat Gearhart, PhD, Laboratory of Molecular Biology, NIA, Baltimore, MD
Myron F. Goodman, PhD, University of Southern California, Los Angeles, CA
Shigenori Iwai, PhD, Osaka University, Osaka, Japan
Tom Kunkel, PhD, Laboratory of Molecular Biology, NIEHS, Research Triangle Park, NC
Christopher Lawrence, PhD, University of Rochester, Rochester, NY
Veronica Maher, PhD, Michigan State University, East Lansing, MI
Stephen West, PhD, Cancer Research UK, London, UK
Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
For further information, contact woodgate@helix.nih.gov.