Translesion synthesis and mutations: On the mutagenic properties of the two DNA lesions, 8-oxo-G and Pt-GG, and the functions of Y-family DNA polymerases and Rev3L on the bypass of each of the DNA lesions in mammalian cells
I studied the capabilities of the two DNA lesions 8-oxo-guanine and cisplatin intrastrand crosslinked 1,2-d(GpG) or Pt-GG to cause mutations in mammalian cells. Using isogenic cell lines generated from mice with selective gene knockouts of distinct DNA polymerases as models, I deduced the biological functions of the translesion DNA polymerases Pol eta, Pol kappa, Pol iota, Rev1 and Rev3L on bypassing each of the lesions 8-oxo-G and Pt-GG. My study takes advantage of the Next Generation Sequencing (NGS) technology to determine mutagenic effects of the DNA lesions in vivo and effects of translesion DNA polymerases on bypassing the lesions. Through adapting a translesion synthesis (TLS) assay into a high-throughput TLS assay which uses NGS, I eliminated steps in the traditional TLS assay that involves E. coli. My study reveals that 8-oxo-guanine caused a high frequency of C->A substitutions, and that significantly, Pol eta correctly bypassed 8-oxo-G in cells. The high mutation rate of 8-oxo-G underlines the importance of DNA repair to remove this lesion before it is replicated. By contrast, Pt-GG caused very few mutations in vivo: the mutation rate of Pt-GG was only two times the background error rate in my experiment. The five translesion DNA polymerases studied bypassed Pt-GG in an error-prone manner; specifically, Rev3L and Rev1 DNA polymerases can each bypass the lesion and Pol iota might share the function of Pol kappa or enhance that of Pol eta to bypass the lesion. Since Pt-GG does not appear to block DNA replication more than 8-oxo-G in mammalian cells, the cytotoxicity of the drug cisplatin that produces the cisplatin-DNA adduct is likely attributed to other causes, such as inter-strand cisplatin-DNA adducts that are more potent for stalling DNA replication.
DNA polymerase, Y-family polymerase, translesion synthesis, DNA lesion, mutation, next generation sequencing
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