Kalam M Abul, Haraguchi Kazuhiro, Chandani Sushil, Loechler Edward L, Moriya Maasaki, Greenberg Marc M, Basu Ashis K
Department of Chemistry, University of Connecticut Storrs, CT 06269, USA.
Nucleic Acids Res. 2006 May 5;34(8):2305-15. doi: 10.1093/nar/gkl099. Print 2006.
Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5'-TGT and 5'-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy.dG is mutagenic inducing primarily targeted Fapy.G-->T transversions. In the 5'-TGT sequence mutational frequency of Fapy.dG was approximately 30%, whereas in the 5'-TGA sequence it was approximately 8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy.dG, though it also exhibited a similar context effect: 4-fold G-->T transversions (24% versus 6%) occurred in the 5'-TGT sequence relative to 5'-TGA. To investigate a possible structural basis for the higher G-->T mutations induced by both lesions when their 3' neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase beta, which is known to incorporate both dCTP (no mutation) and dATP (G-->T substitution) opposite 8-oxo-G. In pol beta, the syn-8-oxo-G:dATP pair showed greater stacking with the 3'-T:A base pair in the 5'-TGT sequence compared with the 3'-A:T in the 5'-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5'-TGT and 5'-TGA sequences. Similarly, syn-Fapy.G:dATP pairing showed greater stacking in the 5'-TGT sequence compared with the 5'-TGA sequence, while stacking for anti-Fapy.G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3'-A:T base pair in the 5'-TGA sequence might cause lower G-->T mutational frequencies in the 5'-TGA sequence compared to 5'-TGT. The corresponding lesions derived from 2'-deoxyadenosine, Fapy.dA and 8-oxo-dA, were not detectably mutagenic in the 5'-TAT sequence, and were only weakly mutagenic (<1%) in the 5'-TAA sequence context, where both lesions induced targeted A-->C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy.G-->T substitutions occur at a higher frequency than 8-oxo-G-->T and that Fapy.dA is very weakly mutagenic, as is 8-oxo-dA.
Fapy.dG和8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代-dG）是由羟基自由基损伤在DNA中形成的。为了研究细胞中这些损伤位点的复制情况，我们构建了一个单链穿梭载体，其在5'-TGT和5'-TGA序列环境中含有损伤位点。修饰后的载体在猴肾（COS-7）细胞中的复制表明，Fapy.dG具有致突变性，主要诱导靶向的Fapy.G→T颠换。在5'-TGT序列中，Fapy.dG的突变频率约为30%，而在5'-TGA序列中约为8%。在平行研究中，发现8-氧代-dG的致突变性略低于Fapy.dG，尽管它也表现出类似的序列环境效应：相对于5'-TGA序列，在5'-TGT序列中发生的G→T颠换增加了4倍（24%对6%）。为了研究当这两种损伤的3'邻位碱基为T时，它们诱导更高频率G→T突变的可能结构基础，我们在DNA聚合酶β的活性位点进行了分子建模研究，已知该酶在8-氧代-G的对面可掺入dCTP（无突变）和dATP（G→T替换）。在DNA聚合酶β中，与5'-TGA序列中的3'-A:T碱基对相比，syn-8-氧代-G:dATP碱基对与5'-TGT序列中的3'-T:A碱基对具有更大的堆积作用，而anti-8-氧代-G:dCTP碱基对在5'-TGT和5'-TGA序列中的堆积作用相似。同样，syn-Fapy.G:dATP碱基对在5'-TGT序列中的堆积作用比在5'-TGA序列中更大，而anti-Fapy.G:dCTP碱基对在这两个序列中的堆积作用相似。因此，对于这两种损伤，与5'-TGA序列相比，损伤:dATP碱基对与5'-TGA序列中的3'-A:T碱基对之间较低的碱基堆积效率可能导致5'-TGA序列中较低的G→T突变频率。源自2'-脱氧腺苷的相应损伤，Fapy.dA和8-氧代-dA，在5'-TAT序列中未检测到致突变性，并且在5'-TAA序列环境中仅具有弱致突变性（<1%），在该序列环境中这两种损伤均诱导靶向的A→C颠换。据我们所知，这是首次使用特异性掺入Fapy.dG或Fapy.dA位点的染色体外探针进行的研究，该研究明确表明在猴肾细胞中，Fapy.G→T替换的发生频率高于8-氧代-G→T，并且Fapy.dA与８-氧代-dA一样具有非常弱的致突变性。
