Nevin Philip, Lu Xueguang, Zhang Ke, Engen John R, Beuning Penny J
Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.
FEBS J. 2015 Jul;282(14):2646-60. doi: 10.1111/febs.13304. Epub 2015 May 11.
Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. Using hydrogen-deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N(2) -furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O(6) -methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.
Y家族DNA聚合酶专门用于复制受损DNA，并且由于其低保真性而与诱变增加有关。据信，Y家族DNA聚合酶选择核苷酸的机制涉及核苷酸转移之前的构象变化，但此类结构变化的实验证据有限。特别是，迄今为止，细菌或真核Y家族DNA聚合酶中核苷酸诱导的构象变化尚未得到广泛表征。利用氢-氘交换质谱法，我们在此证明，在未受损DNA和正确的进入核苷酸存在的情况下，大肠杆菌Y家族DNA聚合酶DinB及其人类直系同源物DNA聚合酶κ会发生保守的核苷酸诱导构象变化。值得注意的是，对于含有N(2)-糠基-脱氧鸟苷的受损DNA也是如此，这两种聚合酶能有效地复制该受损DNA，但对于含有大沟修饰O(6)-甲基-脱氧鸟苷的受损DNA则不然，后者是一种较差的底物。我们的观察结果表明，DinB和DNA聚合酶κ利用一种共同的核苷酸选择机制，该机制涉及由正确的核苷酸和仅优选的DNA底物促进的保守的化学前构象转变。
