Choi Jeong-Yun, Lim Seonhee, Eoff Robert L, Guengerich F Peter
Department of Pharmacology, School of Medicine, Ewha Womans University, 911-1, Mok-6-dong, Yangcheon-gu, Seoul 158-710, Republic of Korea.
J Mol Biol. 2009 Jun 5;389(2):264-74. doi: 10.1016/j.jmb.2009.04.023. Epub 2009 Apr 17.
DNA polymerase (pol) iota, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols eta and kappa, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol iota, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol iota, which was not substantially different from human pols eta and kappa. Opposite template T, only the relative wobble stabilities (inferred from the potential numbers of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota but not the relative W-C or Hoogsteen stabilities, unlike pols eta and kappa. In contrast, opposite C, only the relative W-C stabilities of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota, as with pols eta and kappa. These results suggest that pol iota might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
DNA聚合酶（pol）ι是参与跨损伤DNA合成的哺乳动物Y家族DNA聚合酶成员，此前有研究表明，与利用沃森-克里克（W-C）碱基配对的pol η和κ不同，它特别利用Hoogsteen碱基配对来合成与模板嘌呤相对的DNA。为了研究Hoogsteen、W-C和摆动碱基配对模式在人pol ι选择与模板嘧啶相对的核苷酸中的可能作用，我们对包括7-脱氮嘌呤、肌苷、2-氨基嘌呤、2,6-二氨基嘌呤和6-氯嘌呤在内的修饰嘌呤核苷三磷酸的掺入进行了动力学分析，这些修饰会影响与模板嘧啶相对的碱基对形成中的氢键。嘌呤核苷三磷酸N7原子处的碳取代会破坏Hoogsteen碱基配对，仅轻微抑制pol ι合成与模板嘧啶相对的DNA，这与人类pol η和κ没有实质性差异。与模板T相对时，只有碱基对的相对摆动稳定性（从氢键、空间和静电相互作用的潜在数量推断但未测量）与pol ι掺入核苷酸的相对效率呈正相关，而与相对W-C或Hoogsteen稳定性无关，这与pol η和κ不同。相比之下，与C相对时，只有碱基对的相对W-C稳定性与pol ι掺入核苷酸的相对效率呈正相关，与pol η和κ相同。这些结果表明，pol ι在合成与嘧啶相对的DNA时可能并非不可或缺地需要Hoogsteen碱基配对，而是在选择与T相对的核苷酸时可能更喜欢摆动碱基配对，在与C相对时更喜欢W-C碱基配对。
