Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex

The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.     

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3′→5′ exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3′→5′ exonuclease activity of the hPolε holoenzyme. Together, the 3′→5′ exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.     

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.     

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna^K164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.     

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη''s function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη''s TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη''s function.     

Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight

Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase η (Polη) and polymerase ζ (Polζ), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310–1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Δ, rev3Δ and rev3Δ rad30Δ strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6–4) photoproducts derived from studies with UVC. They further suggest that Polη participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polζ is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polζ, Polη contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.     

Mutator Phenotype Induced by Aberrant Replication

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase δ (polδ) and mutants of Polα, two Polδ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2⁺, which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Δ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Δ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Δ. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.     

Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity.

Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast. RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa. None of the subunits are related through proteolysis or differential phosphorylation. The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA. This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA). In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well. Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule. DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C. RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA. Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus. Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta. These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.     

DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD–primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.     

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.     

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double-stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.     

Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon.

Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase. The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration. A stable complex of RF-C and PCNA on primed single-stranded mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP gamma S. These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex. Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement. DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis. The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast. A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed.     

FF483–484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance

Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF_483–484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.     

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.     

The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

The Human Lagging Strand DNA Polymerase δ Holoenzyme Is Distributive

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5′-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ^AA. The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.     

The fidelity of DNA replication,particularly on GC-rich templates,is reduced by defects of the Fe–S cluster in DNA polymerase δ

Iron-sulfur clusters (4Fe–4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol δ) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol δ are the observations that Pol δ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.     

DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.