Vaisman Alexandra, Takasawa Kohei, Iwai Shigenori, Woodgate Roger
Section on DNA Replication, Repair, and Mutagenesis, Laboratory of Genomic Integrity, Building 6, Room 1A13, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-2725, USA.
DNA Repair (Amst). 2006 Feb 3;5(2):210-8. doi: 10.1016/j.dnarep.2005.09.011. Epub 2005 Nov 2.
Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 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 poleta, where approximately 45% of mutations occur at Ts and approximately 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 poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have 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 two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize 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.
对同步化的野生型S期细胞中紫外线诱导产生的突变谱进行分析发现，只有约25%的突变发生在胸腺嘧啶（T）处，而75%的突变靶向胞嘧啶（C）。在缺乏聚合酶η（polη）的着色性干皮病变种（XP-V）细胞中，突变谱发生了显著变化，约45%的突变发生在T处，约55%发生在C处。目前尚不清楚C→T突变是否实际上代表与C相对的真正错配掺入，或者是否可能是由于在已脱氨为尿嘧啶（U）的紫外线光产物中，腺嘌呤（A）与C正确掺入的结果。为了评估人类聚合酶η在复制绕过此类紫外线光产物中可能发挥的作用（如果有的话），我们在体外分析了聚合酶η依赖的T-U环丁烷嘧啶二聚体（CPD）绕过的效率和保真度。有趣的是，聚合酶η依赖的T-U CPD绕过比相应的T-T CPD更有效。鸟嘌呤（G）与T-U CPD的3'U相对处的错配掺入频率仅比正确的沃森-克里克碱基A低两倍。虽然聚合酶η通常比正确配对的引物更低效地延伸G:3'U-CPD错配，但在相同的测定条件下，聚合酶η依赖的延伸与人类聚合酶η、κ以及酿酒酵母聚合酶ζ观察到的延伸相等或更大。因此，我们推测聚合酶η通过在CPD的3'U相对处频繁错配掺入G来绕过T-U CPD的能力，可能提供了一种机制，通过该机制人类细胞可以降低这些损伤的诱变潜力。
