Active Site Mutations in Mammalian DNA Polymerase δ Alter Accuracy and Replication Fork Progression

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3′ → 5′-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.     

Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in Arabidopsis leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of stn1 and pol α mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in stn1 mutants may represent a manifestation of aberrant replication through telomeres.     

Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

The POL3 encoded catalytic subunit of DNA polymerase δ possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24°, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase δ. SDP5 is most probably the p55 subunit of Polδ of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.     

Aberrant Double-Strand Break Repair Resulting in Half Crossovers in Mutants Defective for Rad51 or the DNA Polymerase δ Complex

Homologous recombination is an error-free mechanism for the repair of DNA double-strand breaks (DSBs). Most DSB repair events occur by gene conversion limiting loss of heterozygosity (LOH) for markers downstream of the site of repair and restricting deleterious chromosome rearrangements. DSBs with only one end available for repair undergo strand invasion into a homologous duplex DNA, followed by replication to the chromosome end (break-induced replication [BIR]), leading to LOH for all markers downstream of the site of strand invasion. Using a transformation-based assay system, we show that most of the apparent BIR events that arise in diploid Saccharomyces cerevisiae rad51Δ mutants are due to half crossovers instead of BIR. These events lead to extensive LOH because one arm of chromosome III is deleted. This outcome is also observed in pol32Δ and pol3-ct mutants, defective for components of the DNA polymerase δ (Pol δ) complex. The half crossovers formed in Pol δ complex mutants show evidence of limited homology-dependent DNA synthesis and are partially Mus81 dependent, suggesting that strand invasion occurs and the stalled intermediate is subsequently cleaved. In contrast to rad51Δ mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion. Thus, the BIR defect observed for rad51 mutants is due to strand invasion failure, whereas the Pol δ complex mutants are proficient for strand invasion but unable to complete extensive tracts of recombination-initiated DNA synthesis.DNA double-strand breaks (DSBs) are potentially lethal lesions that can occur spontaneously during normal cell metabolism, by treatment of cells with DNA-damaging agents, or during programmed recombination processes (54). There are two major pathways to repair DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves the religation of the two ends of the broken chromosome and can occur with high fidelity or be accompanied by a gain or loss of nucleotides at the junction (9). Repair of two-ended DSBs by HR generally occurs by gene conversion resulting from a transfer of information from the intact donor duplex to the broken chromosome (Fig. ​(Fig.1).1). HR occurs preferentially during S and G₂ when a sister chromatid is available to template repair (2, 19, 22). Sister-chromatid recombination events are genetically silent, whereas gene conversion between nonsister chromatids associated with an exchange of flanking markers can result in extensive loss of heterozygosity (LOH) or chromosome rearrangements (3, 21). One-ended DSBs that arise by replication fork collapse or by erosion of uncapped telomeres are thought to repair by strand invasion into homologous duplex DNA followed by replication to the end of the chromosome, a process referred to as break-induced replication (BIR) (35). BIR appears to be suppressed at two-ended breaks, presumably because it can lead to extensive LOH if it occurs between homologues or to chromosome translocations when strand invasion initiates within dispersed repeated sequences (5, 28, 31, 50, 52, 55).Open in a separate windowFIG. 1.Models for gene conversion and BIR. After formation of a DSB, the ends are resected to generate 3′ single-strand DNA tails. One end undergoes Rad51-dependent strand invasion to prime DNA synthesis from the invading 3′ end templated by the donor duplex. For gene conversion by the synthesis-dependent strand annealing model, the extended invading end is displaced and can anneal to the other side of the break; completion of repair requires DNA synthesis primed from the noninvading 3′ end. For a one-ended break, or if the other side of the break lacks homology to the donor duplex, DNA synthesis proceeds to the end of the chromosome. Centromeres are shown as solid ovals and a heterozygous marker centromere distal to the site of repair as A/a.The strand invasion step of BIR is assumed to be the same as that for gene conversion based on the requirement for the same HR proteins: Rad51, Rad52, Rad54, Rad55, and Rad57 (10). However, subsequent steps in BIR are less well defined. Recent studies of the fate of the invading end during BIR in diploid strains with polymorphic chromosome III homologues using a plasmid-based assay have shown that following strand invasion, the invading end is capable of dissociating from the initial homologous template. Following dissociation, the displaced end subsequently reinvades into the same or a different chromosome III homologue by a process termed template switching (52). One of the interesting features of the template switching events is that they occur over a region of about 10 kb downstream of the site of strand invasion and do not extend over the entire left arm of chromosome III. There are a number of possible mechanisms that could account for this apparent change in the processivity of BIR. First, it is possible that the strand invasion intermediate is cleaved by a structure-specific nuclease and once the invading strand is covalently joined to one of the template strands, the strand invasion process is irreversible. Recent studies of Schizosaccharomyces pombe have shown an essential role for Mus81, a structure-specific nuclease, in resolution of sister chromatid recombination intermediates during repair of collapsed replication forks (48). Another possibility is that there could be a switch between a translesion DNA polymerase and a highly processive DNA polymerase during BIR. The translesion polymerases in budding yeast, polymerase ζ (Pol ζ) and Pol η, are encoded by REV3-REV7 and RAD30, respectively (34, 40, 43). Deletion of REV3 has been shown to increase the fidelity of DNA synthesis associated with HR but has no effect on the overall frequency of DSB-induced HR (16). Deletion of POLη in chicken DT40 cells reduces the frequency of DSB-induced gene conversion, and human POL η has been shown to extend the invading 3′ end of D-loop intermediates in vitro (23, 36). However, this same preference for Pol η is not found for Saccharomyces cerevisiae. Instead, DNA synthesis during meiotic and mitotic recombination appears to be carried out by Pol δ, one of the three nuclear replicative polymerases, which normally functions with Pol α in Okazaki fragment synthesis (13, 32, 33, 44). Pol ɛ is thought to be the primary leading-strand polymerase (47), but in the absence of the Pol ɛ catalytic domain, Pol δ is presumed to carry out leading-strand synthesis (24). Recent studies by Lydeard et al. (30) have shown a requirement for the lagging-strand polymerases, Pol δ and Pol α, to form the initial primer extension product during BIR, and Pol ɛ is required to complete replication to the end of the chromosome. In contrast, repair of DSBs by gene conversion does not require Pol α, and there appears to be functional redundancy between Pol δ and Pol ɛ (56).To address the roles of Mus81, Pol δ, and Pol η in BIR and in particular template switching, we used the transformation-based BIR assay with diploids with polymorphic chromosome III homologues. Because the transformation assay can only be used with strains with viable mutations of replication factors, we used a null allele of POL32, encoding a nonessential subunit of the Pol δ complex (14), and a point mutation in the gene encoding the essential catalytic subunit, POL3. The pol3-ct allele results in a truncation removing the last four amino acids of the Pol3 protein; the C-terminal region of Pol3 is implicated in interaction with the other essential subunit of the Pol δ complex, Pol31 (15, 49). The interesting feature of the pol3-ct allele is that it decreases the length of gene conversion tracts during mitotic and meiotic recombination, presumably by affecting the processivity of Pol δ, but confers no apparent defect in normal DNA synthesis (32, 33). Because BIR requires more-extensive tracts of DNA synthesis than gene conversion, we expected the pol3-ct mutant to exhibit a BIR defect. We found that in the absence of a fully functional Pol δ complex, chromosome fragment (CF) formation proceeds by a half-crossover mechanism associated with loss of the template chromosome, an event with potentially catastrophic consequences (6, 57). This was also found to occur in rad51 mutants, suggesting nonreciprocal translocations arise by failure to undergo strand invasion or because replication following strand invasion is inefficient. In contrast to rad51 mutants, the Pol δ complex mutants are proficient for repair of a 238-bp gap by gene conversion and fully resistant to ionizing radiation, suggesting there is a unique requirement for Pol δ to complete BIR. Consistent with studies of gene conversion in S. cerevisiae (33), we found no role for Pol η in BIR or the process of template switching.     

Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants

After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O₈, sus P₃, and T₁₁, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N₇, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q₂₁, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.     

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.     

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.     

Interaction of Loach DNA Polymerase δ with DNA Duplexes with Single-Strand Gaps

The interaction of DNA polymerase purified from eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes with single-strand gaps of 1-13 nucleotides was studied. In the absence of template-restricting DNA, the enzyme elongated primers on single-stranded DNA templates in a distributive manner. However, in the presence of the proximal 5"-terminus restricting the template, the enzyme activity significantly increased. In this case, the enzyme was capable of processive synthesis by filling gaps of 5-9 nucleotides in DNA duplexes. These data indicate that DNA polymerase can interact with both the 3"- and 5"-termini located upstream and downstream from the gap. Analysis of the complexes formed by DNA polymerase and different DNA substrates by electrophoretic mobility shift assay confirmed the assumption that this enzyme can interact with the proximal 5"-terminus restricting the gap. DNA polymerase displayed much higher affinity in duplexes with gaps of approximately 10 nucleotides compared to the standard template–primer complexes. Maximal affinity was observed in experiments with DNA substrates containing unpaired 3"-tails in primers. The results of this study suggest that DNA polymerase exerts high activity in the cell nuclei during repair of DNA intermediates with single strand gaps and unpaired 3"-termini.     

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.     

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.     

Inhibition of Bacteriophage φX174 DNA Replication in dnaB Mutants of Escherichia coli C

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.     

A Role for DNA Polymerase θ in Promoting Replication through Oxidative DNA Lesion,Thymine Glycol,in Human Cells

The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5′ template base, suggests that the polθ active site can accommodate highly distorting DNA lesions.     

Identification of RNF8 as a Ubiquitin Ligase Involved in Targeting the p12 Subunit of DNA Polymerase δ for Degradation in Response to DNA Damage

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8^−/− mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.     

Human DNA Polymerase η Is Required for Common Fragile Site Stability during Unperturbed DNA Replication

Human DNA polymerase η (Pol η) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol η in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol η from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol η-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol ι, the closest relative of Pol η. Our results identify a new role for Pol η in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol η in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.Mutations in the POLH gene that encodes DNA polymerase η (Pol η) are responsible for the variant form of xeroderma pigmentosum (XP-V). XP-V is a rare autosomal recessive disorder characterized by extreme sensitivity to sunlight and a very high incidence of sunlight-induced skin cancer, as are the other forms of “classical” XP (17, 27). However, in contrast to the other nucleotide excision repair (NER)-defective XP complementation groups (XP-A to XP-G), XP-V cells have normal NER but cannot support translesion synthesis (TLS) past DNA-containing cyclobutane pyrimidine dimers (CPDs) (27). Purified Pol η, the TLS polymerase that is mutated in XP-V, is able to synthesize past this lesion with a high level of efficiency (28), and in a majority of cases it inserts the correct nucleotide, adenine, opposite the two thymines contained in the cyclobutane pyrimidine dimer ring (26).The ability to replicate efficiently past UV pyrimidine dimers has been the principal—or sole—function assigned thus far to Pol η. In the absence of Pol η, cells display an increased rate of UV-induced mutagenesis and carcinogenesis (23) that may reflect inefficient or error-prone synthesis by another polymerase. In mouse cells, this back-up polymerase may be Pol ι (12). Despite its ability to replicate past cyclobutane pyrimidine dimers, Pol η does not appear to be able to carry out TLS past the other major UV photoproduct, the pyrimidine (6-4) pyrimidone photoproduct [(6-4)PP] in vitro or in vivo. It can, however, replicate past a limited number of other types of DNA damage in vitro, albeit with a lower level of efficiency than past CPDs (21). Whether the bypass of these lesions is performed in vivo by Pol η is less clear. For example, XP-V cells are sensitive to cisplatin, suggesting that bypass of cisplatin lesions may depend on Pol η (1). Combined NER- and Pol η-mediated lesion bypass has also been suggested as the likely mechanism for repairing DNA interstrand cross-links formed by mitomycin C (46) and psoralen (32). In contrast, Pol η does not appear to play a role in replication past endogenous lesions such as 8-oxoguanine (3) or abasic sites (2).It has been difficult to visualize or identify sites of action of Pol η or any of the other TLS polymerases by immunofluorescence due to their low levels of expression. However, in cells that mildly overexpress Pol η, it has been possible to localize the polymerase to nuclear replication factories during S phase. This localization depends on several motifs located close to the C terminus of Pol η, including an NLS and a ubiquitin-binding zinc finger domain (7, 18). Localization of Pol η in replication factories may concentrate the polymerase near sites of replication to facilitate recruitment to carry out TLS. If cells cannot remove or synthesize through a lesion blocking the replication fork, then homology-dependent recombinational repair (HRR) may be used to restart the replication fork (11, 34). RAD51-mediated HRR has been shown to be important for the repair of DNA damage during replication in all organisms (20, 31, 42). Recent evidence has suggested that Pol η, in addition to its role in TLS, may participate in HRR. This has been suggested by analyses of gene conversion in chicken DT40 cells during immunoglobulin gene diversification (19), as well as by in vitro experiments showing that Pol η is capable of promoting extension of the invading strand in D-loop structures to facilitate RAD52-mediated second-end capture during recombination-mediated repair (29, 30). The functional importance of this observation is less clear. Recent evidence from yeast argues that the bulk of heteroduplex DNA strand extension during HRR is mediated by the preferential recruitment of a replicative DNA polymerase, Pol δ (25). Moreover, there is no obvious recombination deficit in XP-V patients or in XP-V cells beyond a modest elevation in the frequency of UV-induced sister chromatid exchanges (10).In order to better understand the functional roles and importance of Pol η in human cells, we used short hairpin RNAs (shRNAs) to selectively deplete Pol η from cells and then determined how the loss of Pol η affected cell cycle progression, DNA replication dynamics, and cell proliferation in otherwise unperturbed cells. These experiments revealed an unexpected role for Pol η in maintaining chromosomal stability and preventing common fragile site (CFS) breakage during unperturbed S phase. Our results thus broaden the functional role of Pol η in human cells to include the maintenance of genomic stability during unperturbed DNA replication in S phase.