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 xeroderma pigmentosum; 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 kingdoms of life. ? ? Our studies in Escherichia coli centered on polV?s ability to facilitate TLS and were performed as part of a collaborative study with Myron Goodman at the University of Southern California. In particular, we investigated the nature of the interactions between RecA and polV. It had previously been assumed that RecA binds to the damaged DNA template strand being copied by polV. Remarkably, however, our recent studies revealed that polV-catalyzed translesion synthesis, in the presence or absence of the beta-processivity-clamp, occurs only when RecA nucleoprotein filaments assemble on separate single-stranded (ss)DNA molecules in trans. A 3'-proximal RecA filament end on trans DNA is essential for stimulation and is strengthened by further polV-RecA interactions occurring elsewhere along a trans nucleoprotein filament. Based upon these observations, we suggested that trans-stimulation of polV by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis on a damaged template strand obstructing translesion DNA synthesis, despite the absolute requirement of RecA for SOS mutagenesis.? ? As part of our studies in Archaea, we identified and characterized five novel thermostable Dpo4-like enzymes, as well as two recombinant chimeras that have enhanced enzymatic properties compared to the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR, and can bypass DNA lesions that normally block Taq. By using a blend of Taq and Dpo4 enzymes, we obtained a PCR amplicon from UV-irradiated DNA that was unamplifyable with Taq alone. We hypothesize that the inclusion of thermostable Dpo4-like polymerases in PCR reactions will therefore augment the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications. ? ? Studies on human human DNA polymerases eta and iota focussed on how cells regulate the low-fidelity polymerases so as to minimize their inadvertent access to primer-termini. As part of these studies, we discoverd that one mechanism employed by human cells relies on a specific and direct interaction between DNA polymerases iota and eta with ubiquitin (Ub). Indeed, we showed that both polymerases interact noncovalently with free polyUb chains, as well as mono-ubiquitinated proliferating cell nuclear antigen (Ub-PCNA). Mutants of pol iota (P692R) and pol eta (H654A) were isolated that were defective in their interactions with polyUb and Ub-PCNA, whilst retaining their ability to interact with unmodified PCNA. Interestingly, the polymerase mutants exhibited significantly lower levels of replication foci in response to DNA damage, thereby highlighting the biological importance of the polymerase-Ub interaction in regulating the access of the TLS polymerases to stalled replication forks in vivo.? ? In vitro studies with human DNA polymerase iota focused on understanding its role in the bypass of UV-induced photoproducts. Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type human S-phase cells reveals that only ~25% of mutations occur at Thymine (T), whilst 75% are targeted to Cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of the major human TLS enzyme, pol eta, where ~45% of mutations occur at Ts and ~55% at Cs. At the present time, it is unclear whether the C->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of Adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to Uracil (U). In order to assess the role that human pol iota might play in the replicative bypass of such UV-photoproducts, we analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurred more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only 2-fold less frequently than the correct Watson-Crick base, A. Thus, based upon our in vitro observations, we hypothesized that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.
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