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 kingdoms of life.? ? Our studies in Escherichia coli centered on polV(R391) encoded by rumA'B from the IncJ conjugative-transposon, R391. Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. Interestingly, when expressed in a delta umuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively. Analysis of the spectrum of polV(R391)-dependent mutations in rpoB revealed a unique genetic fingerprint that was typified by an increase in C:G ->A:T and A:T->T:A transversions at certain mutagenic hot-spots. Biochemical characterization of polV(R391) highlighted the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot-spots. Purified polV(R391) can also bypassed a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3'T of the dimer than opposite an undamaged T. Our study therefore provided evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA'B, as well as explained its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo. ? ? As part of our studies in Archaea, we generated mutant variants of Taq DNA polymerase that could bypass a variety of DNA lesions in vitro. It is well known that in the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological, or forensic interest is inevitably damaged. By using compartmentalized self replication of polymerase genes from the genus Thermus, we evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000 years old cave bear DNA, they out-performed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions, where Taq no longer yielded a product. Our results demonstrated that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archaeology and forensic medicine.? ? Studies on human human DNA polymerases iota focused on it enzymatic properties in vitro and in vivo. For example, all DNA polymerases require a divalent action for catalytic activity and it is generally assumed that Mg is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn in vitro. Surprisingly, we found that human DNA polymerase iota also exhibited the greatest activity in the presence of 0.05-0.2 Mn. Peak activity in the presence of Mg was observed in the range of 0.1-0.5 mM, and was significantly reduced at concentrations > 2mM. Steady-state kinetic analyses revealed that Mn increases the catalytic activity of pol iota by 30 to 60,000-fold through a dramatic decrease in the Km for nucleotide incorporation. Interestingly, while pol iota preferentially misinserted G opposite T by a factor of 1.4 to 2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn. Low levels of Mn also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro, including a UV-induced cyclobutane pyrimidine dimer, where the bypass efficiency increase from just 2% in the presence of Mg to 60% in the presence of Mn. Titration experiments revealed a strong preference of pol iota for Mn even when Mg was present in a >10-fold excess. Our observations therefore raised the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn rather than Mg, as has been tacitly assumed. ? ? Our observations that in the presence of Mn, pol iota can bypass UV-induced cyclobutane pyrimidine dimers relatively efficiently, may be of significant biological interest, particularly in humans with the Xeroderma Pigmentosum Variant (XP-V) phenotype. XP-V 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 that has a very unusual spectrum compared to normal cells. The primary defect in XP-V cells is lack of functional DNA polymerase eta, which normally protects humans from UV-induced mutations through its ability to readily insert adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XP-V cells strongly suggest that in the absence of polymerase eta, an abnormally error-prone polymerase substitutes. Indeed, our in vitro replication studies on polymerase iota demonstrated that it readily replicates past 5T-T3 and 5T-U3 cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3 nucleotide. To test the hypothesis that pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XP-V cells, we identified an unlimited lifespan XP-V cell line that had approximately half the expression levels of pol iota and compared the parental cells and derivatives for their frequency and spectrum of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV, and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to ≤50% that of the parent. Our data therefore supports the hypothesis that in XP-V cells lacking pol eta, pol iota is responsible for the high frequency and abnormal spectrum of UV-induced mutations, and ultimately their malignant transformation.
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