Rajat S. Das

(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

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.

Structures of (5'S)-8,5'-Cyclo-2'-deoxyguanosine Mismatched with dA or dT.

Diastereomeric 8,5′-cyclopurine 2′-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base, are induced in DNA by ionizing radiation. They are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients. If not repaired, the S-8,5′-cyclo-2′-deoxyguanosine lesion (S-cdG) induces Pol V-dependent mutations at a frequency of 34% in Escherichia coli. Most are S-cdG → A transitions, suggesting mis-incorporation of dTTP opposite the lesion during replication bypass, although low levels of S-cdG → T transversions, arising from mis-incorporation of dATP, are also observed. We report the structures of 5′-d(GTGCXTGTTTGT)-3′·5′-d(ACAAACAYGCAC)-3′, where X denotes S-cdG and Y denotes either dA or dT, corresponding to the situation following mis-insertion of either dTTP or dATP opposite the S-cdG lesion. The S-cdG·dT mismatch pair adopts a wobble base pairing. This provides a plausible rationale for the S-cdG → A transitions. The S-cdG·dA mismatch pair differs in conformation from the dG·dA mismatch pair. For the S-cdG·dA mismatch pair, both S-cdG and dA intercalate, but no hydrogen bonding is observed between S-cdG and dA. This is consistent with the lower levels of S-cdG → T transitions in E. coli.

Abstract 1328: Replication inhibition and mutagenicity of 8,5’-cyclopurine-2’-deoxynucleosides in Escherichia coli

8,5’-Cyclopurine-2’-deoxynucleosides are unique tandem DNA lesions formed by oxidation or ionizing radiation, which are repaired only by nucleotide excision repair. They have been suspected to be responsible for neurodegeneration and other diseases. But their replication properties are little studied. In the current work, we have evaluated replication of a pMS2 plasmid containing a single (59s)-8,5’-cyclo-2’-deoxyadenosine (59s-cdA) or (59s)-8,5’-cyclo-2’-deoxyguanosine (59s-cdG) at a preselected site in Escherichia coli. In the wild type E. coli cells, viability of 59s-cdA and 59s-cdG plasmids dropped to approximately 1% and 0.5%, respectively, relative to control. The viability of each was further reduced in pol II-deficient strain, but it was most pronounced in a pol V-deficient strain, where it dropped ten-fold. By contrast, viability increased 4-7-fold in a pol IV-deficient strain. Upon induction of SOS functions, viability increased ten-fold in the wild type strain. Though less pronounced, viability increased in the other strains as well. We conclude that pol V is necessary for translesion synthesis of the cyclopurines and that it may work cooperatively with pol II, which is also needed for bypass. In addition, it appears that although pol IV competes with the other bypass polymerases, it is inefficient in bypassing these lesions. In the absence of SOS, 59s-cdA-to-G mutations occurred at a low frequency, whereas 59s-cdG-to-A events were detected at a much higher frequency. With SOS, 59s-cdA-to-T and 59s-cdG-to-T were also detected. [Supported by NIEHS grant ES-013324] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1328. doi:10.1158/1538-7445.AM2011-1328