Hashim M F, Marnett L J
A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Center in Molecular Toxicology and the Vanderbilt Cancer Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 1996 Apr 12;271(15):9160-5. doi: 10.1074/jbc.271.15.9160.
Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (C-PdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10(3) - 10(4)-fold compared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the Vmax/Kmvalues for incorporation of dGMP were similar in both sequence contexts, whereas the Vmax/Km for dAMP incorporation increased 4.7-fold when the base pair 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1- and 2-base deletions were major products of replication in both sequence contexts. Full-length products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation of dAMP opposite PdG. The observation of PdG --> T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter.
包含丙炔脱氧鸟苷（PdG）的模板引物在两种不同的序列环境（C-PdG-C和T-PdG-T）中由DNA聚合酶I的Klenow片段进行复制。与未修饰的模板引物相比，模板链中PdG的存在使体外DNA合成的程度降低了10³ - 10⁴倍。在加合物3'端1个碱基处和加合物相对位置观察到部分阻断。嘌呤优先掺入到加合物相对位置；在两种序列环境中，dGMP掺入的Vmax/Km值相似，而当PdG 3'端的碱基对从C:G变为T:A时，dAMP掺入的Vmax/Km增加了4.7倍。在两种序列环境中，包含1个和2个碱基缺失的寡核苷酸都是复制的主要产物。在含有T-PdG-T的模板中观察到了全长产物，但在含有C-PdG-C的模板中未观察到。主要的全长产物是由dAMP残基掺入到PdG相对位置导致的。动力学分析表明，全长产物中dAMP选择性掺入的主要因素是含有PdG:dA末端的模板引物的优先延伸，而不是dAMP优先掺入到PdG相对位置。在体外DNA复制过程中，在T-PdG-T环境中观察到了PdG→T突变，而在C-PdG-C环境中未观察到，这与体内实验的结果相似，即在前者序列环境中PdG会诱导碱基对替换，而在后者中则不会。
