Harris Victoria H, Smith Clifford L, Jonathan Cummins W, Hamilton Alan L, Adams Harry, Dickman Mark, Hornby David P, Williams David M
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
J Mol Biol. 2003 Mar 7;326(5):1389-401. doi: 10.1016/s0022-2836(03)00051-2.
The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.
核苷类似物dP（6 - （2 - 脱氧 - β - D - 呋喃核糖基） - 3,4 - 二氢 - 6H,8H - 嘧啶并[4,5 - c][1,2]恶嗪 - 2 - 酮）具有矛盾的氢键特性，其中P的亚氨基互变异构体可与腺嘌呤形成碱基对，其氨基互变异构体可与鸟嘌呤形成碱基对。已合成6 - 甲基 - 3,4 - 二氢 - 6H,8H - 嘧啶并[4,5 - c][1,2]恶嗪 - 2 - 酮（N - 甲基P）的固定亚氨基和氨基互变异构体，并通过X射线晶体学获得其结构。N - 甲基P的互变异构常数已根据固定互变异构体的pK(a)值计算得出，并且已确定其5'-三磷酸（dPTP）被DNA聚合酶I的无外切核酸酶的Klenow片段掺入的动力学参数。发现互变异构常数与dPTP的掺入特异性之间存在很强的相关性。这些结果支持了天然碱基的次要互变异构形式可能在DNA复制期间的取代诱变中起重要作用的提议。此外，它们暗示DNA聚合酶在核苷酸掺入过程中对碱基对施加特定的空间要求。
