Trincao Jose, Johnson Robert E, Wolfle William T, Escalante Carlos R, Prakash Satya, Prakash Louise, Aggarwal Aneel K
Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, New York, New York 10029, USA.
Nat Struct Mol Biol. 2004 May;11(5):457-62. doi: 10.1038/nsmb755. Epub 2004 Apr 11.
The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G.T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G.T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G.T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide.
DNA聚合酶延伸错配碱基的能力与否直接影响基因组突变的产生。我们在此报告了对来自嗜热栖热菌的Y家族聚合酶Dpo4从与模板G或T相对的所有错配碱基处延伸的能力的动力学分析。Dpo4延伸这些错配碱基的效率同样低下，令人惊讶的是，其中包括一个预计与沃森-克里克碱基配对几何结构紧密相符的G.T错配。为阐明其原因，我们解析了在有即将掺入的核苷酸存在的情况下，与G.T错配引物模板结合的Dpo4的结构。作为对照，我们还确定了与引物末端匹配的A-T碱基对结合的Dpo4的结构。这些结构为Dpo4延伸G.T错配效率低下提供了一个依据：一种反向摆动使引物3'-OH偏离即将掺入的核苷酸。
