DNA Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent of Error-Prone Synthesis, and the Role of dNTP Pools

Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. Cells utilize specialized low-fidelity Translesion Synthesis (TLS) DNA polymerases to bypass lesions and rescue arrested replication forks. TLS is a highly conserved two-step process that involves insertion of a nucleotide opposite a lesion and extension of the resulting aberrant primer terminus. The first step can be performed by both replicative and TLS DNA polymerases and, because of non-instructive DNA lesions, often results in a nucleotide misincorporation. The second step is almost exclusively catalyzed by DNA polymerase ζ (Polζ). This unique role of Polζ allows the misincorporated nucleotide to remain in DNA, resulting in a mutation. Because of the low fidelity of Polζ, a processive copying of undamaged DNA beyond the lesion site by this polymerase is expected to be mutagenic. To restore faithful DNA replication, Polζ must be immediately replaced by an accurate replicative DNA polymerase. However, in vivo evidence for this is lacking. To elucidate the late steps of TLS, we aimed to determine the extent of error-prone synthesis associated with mutagenic lesion bypass in yeast. We demonstrate that TLS tracts can span up to 1,000 nucleotides after lesion bypass is completed, leading to more than a 300,000-fold increase in mutagenesis in this region. We describe a model explaining how the length of the error-prone synthesis may be regulated and speculate that Polζ could contribute to localized hypermutagenesis, a phenomenon that plays an important role in cancer development, immunity and adaptation. To gain further insight into the mechanisms of Polζ -dependent mutagenesis, we determined how the increase in dNTP levels occurring in response to DNA damage in yeast affects Polζ function. Surprisingly, increasing the dNTP concentrations to “damage-response” levels only minimally affected the activity, fidelity and error specificity of Polζ, suggesting that, unlike the replicative DNA polymerases, Polζ is resistant to fluctuations in the dNTP levels. Importantly, we demonstrated that Polζ -dependent mutagenesis in vivo does not require high dNTP levels either. Altogether, our results suggest a novel function of Polζ in bypassing lesions or other impediments when dNTP supply is limited

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements. Mitotic entry before completion of DNA replication causes genome instability via an unknown mechanism. Using Xenopus egg extracts, Deng et al. find that mitotic cyclin-dependent kinase triggers replication fork breakage and DNA rearrangements. The mechanism requires TRAIP-dependent ubiquitylation of the replicative helicase followed by p97 ATPase-dependent helicase removal from chromatin.</p

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

TRAIP is a master regulator of DNA interstrand crosslink repair

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2–7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles

Participation of DNA Polymerase ζ in Replication of Undamaged DNA in Saccharomyces cerevisiae

Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase ζ (Polζ) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due to mutations in replication genes. Here we present evidence that this defective-replisome-induced mutagenesis (DRIM) results from the participation of Polζ in the copying of undamaged DNA rather than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways. Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM reveals the characteristic error signature seen during replication of undamaged DNA by Polζ in vitro. These results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication inhibitor hydroxyurea causes a Polζ-dependent increase in mutagenesis. This suggests that DRIM represents a response to replication impediment per se rather than to specific defects in the replisome components

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

<div><p>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 <i>in vivo</i> 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.</p></div

UV lesion bypass is associated with increased mutagenesis downstream of the lesion.

<p>The position of the presumed UV lesion at the <i>ura3-G764A</i> mutation site is indicated in red. Distribution of untargeted mutations in UV-induced Ura⁺ revertants is shown above the horizontal scale bar. Each vertical line represents a single mutation. Mutations found within 1000 bp downstream from the lesion are in black, those in other regions are in grey. Blue lines below the horizontal scale bar represent mutations found in UV-induced Can^r mutants of the <i>ura3-G764A</i> strain. The data are based on DNA sequence analysis of 165 independent Ura⁺ revertants and 161 independent Can^r mutant. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 1-kb region between Ura⁺ revertants and Can^r controls.</p

THF bypass is associated with increased mutagenesis downstream of the lesion.

<p>(A) Distribution of mutations found in the products of TLS through the THF lesion. The THF position is indicated in red. Each vertical line represents a single mutation; the mutation found twice is marked with the asterisk. Mutations in the TLS products within 220 bp from the lesion are in black, other mutations in TLS products are in grey. Blue lines below the horizontal scale bar represent mutations found in the control substrates without the THF. The data are based on DNA sequence analysis of 394 THF bypass products and 456 products of the control plasmid replication. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 220-nucleotide region between TLS products and control plasmids. (B) Types of mutations observed in the THF bypass products and control plasmids. C → T changes are shown for the transcribed strand that is exposed as ssDNA during the plasmid construction. The double asterisk indicates a statistically significant difference (p = 0.0347, Fisher’s exact test).</p