Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
Biochemistry. 2011 May 17;50(19):3862-5. doi: 10.1021/bi2004944. Epub 2011 Apr 25.
8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.
8,5'-环嘌呤构成了一类重要的电离辐射诱导的串联 DNA 损伤，只能通过核苷酸切除修复（NER）来修复。它们在 NER 受损的细胞中积累，如 Cockayne 综合征 B 型和某些着色性干皮病患者。一个包含（5'S）-8,5'-环-2'-脱氧鸟苷（S-cdG）的质粒在特定的 DNA 聚合酶敲除的大肠杆菌中复制。野生型菌株的存活率<1%，但在 SOS 下增加到 5.5%。在 pol II(-)菌株中存活率进一步下降，而在 pol IV(-)菌株中则显著增加。值得注意的是，pol V(-)菌株中没有回收后代，表明 pol V 绝对需要绕过 S-cdG。后代分析表明，S-cdG 具有显著的诱变作用，在 SOS 下诱导约 34%的突变。大多数突变是 S-cdG→A 突变，尽管也发生了 S-cdG→T 突变和 5'C 的缺失。UvrABC 核酸内切酶在双链 51 -mer 上对 S-cdG、S-cdA 和 C8-dG-AP 的切割表明，切割率为 C8-dG-AP>S-cdA>S-cdG。总之，S-cdG 是 DNA 复制的主要障碍，具有高度诱变作用，在大肠杆菌中修复缓慢。
