DNA Replication, Repair, and Mutagenesis In Eukaryotic A

Child Health And Human Development

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Abstract

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. In Escherichia coli, studies centered on polIV and polV and their ability to facilitate TLS. In the past year, we discovered that that pol V physically interacts with the cell?s main recombinase, RecA, through two distinguishable mechanisms. The first occurs when Pol V binds to RecA through its UmuC subunit in the absence of DNA and ATP, whilst the second occurs through its UmuD? subunit in the presence of DNA and ATP, but not ATP hydrolysis. Notably, pol V fails to synthesize DNA with a RecA mutant (RecA1730) that is defective in promoting SOS mutagenesis in vivo, suggesting that RecA serves as an obligate pol V accessory factor, whose principal role is to activate pol V for SOS mutagenesis. Although the their principal biological roles of polIV and polV appear to involve TLS and neither enzyme is at the present time known to be involved in base excision repair (BER), we nevertheless observed apurinic/apyrimidinc 5?-deoxyribose phosphate (AP/5?-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3?-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5?-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase _eta, which is a key enzyme used in short patch BER and suggests that both pols IV and V may participate in a hitherto unidentified BER pathway in E.coli. Scientists in the LGI previously identified, cloned, and characterized a DinB homolog from the archaeon Sulfolobus solfataricus P2, called DNA polymerase IV (Dpo4). In a collaborative study, researchers crystallized the enzyme and solved by X-ray crystallography the structure of ternary complexes of the polymerase together with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggested two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. Interestingly, the correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not. In the past year, 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 DNA polymerase iota focused on understanding the role of the polymerase 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 occurs 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.