Participation of mouse DNA polymerases iota, eta, and rev1 in translesion synthesis of carcinogen induced DNA adducts and carcinogenesis.

Recent advances in understanding the molecular mechanisms of mutagenesis indicate that most mutations are dependent on the activity of translesion synthesis DNA polymerases. The impact of reducing the level of these polymerases on mutagenesis and carcinogenesis in mouse models is poorly defined. Using knock out strategies we were able to remove polymerase eta (pol ç), and polymerase iota (pol é) and pol eta/pol iota from the mouse and lower REV1 in the mouse lung. This dissertation reports the changes in UV-induced carcinogenesis and mutagenesis that were observed. Mutagenesis data in pol iota deficient cells clearly indicate pol iota as a mutagenic TLS polymerase in UV lesion bypass. Pol iota removal effectively lowered the mutational frequency in both pol eta null and wild-type backgrounds. Unexpectedly, after Hprt mutant screening, pol eta and pol iota deficient cells were found to participate in UV lesion bypass in a strand-specific manner. This suggests that not only does bypass of photoproducts occur but occurs in an asymmetrical fashion, with preference of polymerases for leading or lagging strand. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we examined how pol iota status contributes to carcinogenesis. Despite the fact that pol iota was mutagenic in bypass of UV induced lesions, its removal accelerated cancer formation in the pol eta null background. The mechanisms behind this tumor suppressor function remain elusive, but indicate pol iota may have additional cellular roles in conjunction with its polymerase activity. Examining the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we developed strategies to reduce REV1 in mouse models of lung carcinogenesis. Endogenous REV1 mRNA in the lung was effectively lowered with the use of gene delivery of REV1 targeting ribozyme. This reduction was found to effectively decrease the multiplicity of B[a]P-induced lung tumors. This reduction did not affect the size or types of tumors induced suggesting inhibition of cancer formation occurred at the initiation step. Collectively, these data yield insight into the molecular mechanisms of mutagenesis that induce cancer formation

Unconventional Ubiquitin Recognition by the Ubiquitin-Binding Motif within the Y-Family DNA Polymerases ι and Rev1

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) ι and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol ι and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol ι contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol ι in replication foci and Rev1-mediated DNA damage tolerance in vivo.National Institute of General Medical Sciences (U.S.) (Grant GM-079376)American Cancer Society. Research ProfessorshipNational Institute of Environmental Health Sciences (Grant P30 ES-002109

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts

DNA Polymerase Eta Participates in the Mutagenic Bypass of Adducts Induced by Benzo[a]pyrene Diol Epoxide in Mammalian Cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers