(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

Abstract

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.

Scheme 1. Structures of S-cdA and S-cdG

Figure 1

Incisions of 51-mers containing C8-dG-AP (top left), S-cdG (top right), S-cdA (bottom left), and control (or no damage) (bottom right). The lesion-containing and control strands were 5′-terminally labeled with ³²P. The DNA substrates were incubated with UvrABC proteins in UvrABC buffer in the presence of 1 mM ATP at 37 °C for the indicated periods. The reaction products were analyzed via 12% urea–PAGE under denaturing conditions.

Figure 2

Kinetics of UvrABC incisions of substrates containing C8-dG-AP (●), S-cdG (◼), and S-cdA (▲).