Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. It is now known that many of the proteins long implicated in the mutagenic process are, in fact, low-fidelity DNA polymerases that can replicate by traversing damaged DNA in a process termed translesion DNA synthesis (TLS). Humans with defects in one such polymerase, pol eta, are afflicted with the Xeroderma pigmentosum syndrome; they exhibit sensitivity to ultraviolet light and are prone to sunlight-induced skin cancers.? ? In the past year, experiments aimed at understanding the functions of Y-family polymerases spanned the evolutionary spectrum and included studies on organisms from all three domains of life. ? ? Escherichia coli possesses five known DNA polymerases (pols) and their multiplicity requires that their access to genomic DNA must be exquisitely regulated. To gain insights into the interplay that takes place amongst E.colis five DNA polymerases, we exploited the fact that each polymerase leaves a distinct genetic fingerprint when copying DNA. We analyzed the spectrum of rpoB mutants generated in a set of isogenic strains carrying a mutation in one, or more, of E.colis chromosomally encoded DNA polymerases, as well as in 3 related strains that moderately overexpressed polV-like polymerases. The spectrum of mutations was dominated by AT to GC and CG to TA transitions at four well-defined hot-spots. These mutations were not dependent upon pols II, IV or V, as a considerable number of mutants were recovered at the hot-spots in the absence of each polymerase. However, it appears that all three polymerases can modulate the extent of mutagenesis at these sites, since the magnitude of mutagenesis varied considerably in the polymerase-deficient strains. In contrast, in a strain lacking polA there was a striking reduction in transition mutagenesis, suggesting that these mutagenic events are largely dependent upon pol I. Our study also revealed that transversion mutations are largely dependent upon the combined actions of pol IV and pol V and that the cellular levels of pol V are limiting for transversion mutagenesis. Furthermore, and in contrast to pols I, IV and V which promote mutagenesis, pol II appears to play a largely antimutagenic role by suppressing mutations at certain hot-spots. Until now, it has tacitly been assumed that spontaneous mutations occurring on the E.coli genome largely arise during genome duplication performed by the cells replicase, pol III. However, our observations indicate that there is, in fact, considerable switching amongst E.colis five DNA polymerases in the absence of exogenous DNA damage and that such interplay modulates the extent of spontaneous mutagenesis occurring on the E.coli chromosome.? ? As part of a collaborative study with Stephen D. Bell (University of Oxford, UK), we assayed the effect of archaeal PCNA on the processivity of Dpo4, as well as investigated the mechanism whereby circular PCNA is loaded onto DNA. Unlike eukaryotic PCNA, which is comprised of a homotrimer of PCNA protomers, Sulfolobus solfataricus PCNA is comprised of a heterotrimer of three discrete PCNA subunits (PCNA1, PCNA2 and PCNA3). We first confirmed that the processivity of Dpo4 is greatly stimulated in the presence of heterotrimeric PCNA1/2/3, and that Dpo4 specifically interacts with the PCNA1 subunit and shows little, to no affinity, for PCNA2 or PCNA3 subunits. To test how many interfaces within PCNA need to be opened during the clamp loading process, we exploited the unique asymmetry of S. solfataricus PCNA. Our strategy was to covalently fuse PCNA subunits by the use of linker peptide sequences to join the N-terminus of one PCNA subunit to the C-terminus of its neighbor, thereby covalently sealing inter-subunit interfaces. Employing these constructs in clamp loading assays allowed us to determine that productive loading of PCNA only requires opening of a single inter-subunit interface; specifically, that between PCNA3 and PCNA1. ? ? It is well known that DNA is subject to a variety of chemical modifications that alter its structure. One such modification is DNA methylation, which can be caused by endogenous chemicals, products of metabolism, environmental exposure, or treatment with several cancer chemotherapeutics. The major products in DNA exposed to SN2 methylating agents are N7-methylguanine and N3-methyladenine (3MeA). 3MeA accounts for approximately 20% of the base damage formed by SN2 methylating agents and is considered to be the major cytotoxic lesion produced by such chemicals. However, it has been extremely difficult to prove that 3MeA blocks replication directly, as the half-life of 3MeA in vitro is estimated to be between 12 and 24 hours, thereby precluding biochemical analysis. To circumvent these problems, we synthesized a stable 3-deaza analog of the nucleoside 3-methyl-2-deoxyadenosine (3dMeA) that can be incorporated into synthetic oligonucleotides that we have used as templates for DNA replication in vitro. As expected, the 3dMeA lesion blocked both human DNA polymerases alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as S. cerevisiae pol eta were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we demonstrated that in S. cerevisiae lacking 3MeA repair, human pol eta, pol iota and especially pol kappa are capable of restoring methyl methanesulfonate (MMS)-resistance to the normally MMS-sensitive strain. ? ? In a collaborative study with Samuel Wilson (NIEHS), we investigated the role of DNA polymerase iota in BER. Mouse embryonic fibroblasts (MEFs) deficient in pol beta demonstrate increased sensitivity to alkylating agents such as methyl methanesulfonate (MMS), yet cellular extracts from pol beta null cells have some residual BER activity, suggesting that other polymerases could substitute for pol beta in BER. To directly test if pol iota might possibly function as a back up BER enzyme in pol beta null cells, we determined the MMS-sensitivity of MEFs deficient in pol beta and iota. Interestingly, MEFs that were doubly deficient in pol beta and pol iota were more sensitive to the killing effects of MMS than MEFs deficient in pol beta alone, suggesting that pol iota may indeed participate in the BER of MMS induced lesions. However, expression of mouse pol iota in the pol beta-deficient and pol iota-deficient cells did not affect the resistance of these cells to the alkylating agent MMS. Thus, it appears that the difference in the MMS-sensitivity of the pol iota-deficient and -proficient cell lines is likely to be a result of uncharacterized differences between the two cell lines. As a consequence, we conclude that although pol iota possesses dRP lyase activity, expression of the polymerase has negligible impact on the alkylation damage sensitivity of cells lacking pol beta.? ? Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 20-fold higher than normal frequency of UV-induced mutations and a very unusual spectrum of mutations. The primary defect in XP-V cells is a lack of functional pol eta. In a collaborative study with Kenneth Kraemer (NCI), we surveyed the molecular basis of XP-V worldwide, by measuring levels of pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Subsequent DNA sequencing identified 10 different POLH mutations. Nine of these mutations were located in the catalytic domain of the polymerase, and one in the C-terminus of the protein involved in mediating protein-protein interactions between pol eta and its partners.
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