Title

In vitro and In vivo Mutagenesis Studies of Gamma-Radiation-Induced Guanine Thymine Intra-strand Crosslink

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Genetics|Chemistry, Biochemistry|Health Sciences, Oncology

Degree

Ph.D.

Abstract

γ-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replication in vitro and is mutagenic in mammalian cells. Herein we report in vitro translesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase η (hPol η and yPol η). dAMP misincorporation opposite the cross-linked G by yPol η was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for G:A compared to G:C. For hPol η, both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-hased DNA sequencing assay, which showed that yPol η was more error-prone. Mutational frequencies of yPol η and hPol η were 36% and 14%, respectively. Targeted G→T was the dominant mutation by both DNA polymerases. But yPol η induced targeted G→T in 23% frequency relative to 4% by hPol η. For yPol η, targeted G→T and G→C, constituted 83% of the mutations. By contrast, with hPol η, semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol η showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.^ Next, the nucleotide incorporation opposite the cross-linked bases, as well as further extension by dinB homologs, human DNA polymerase K and Dpo4 was investigated. It was observed that catalytic efficiency of dCMP incorporation by Dpo4 decreased by ∼1100 fold opposite cross-linked G relative to control. The dCMP incorporation, followed by extension to the next base, was reduced nearly 2900-fold. In the case of hpol κ, catalytic efficiency of dCMP incorporation decreased by approximately 500-fold opposite the cross-linked G, relative to the control, whereas dCMP incorporation followed by extension was reduced by 38,000-fold. The above-mentioned plasmid based assay was used to analyze the full-length extension products of Dpo4. Mutational frequency of Dpo4 was 9.5% for the G[8,5-Me]T cross-link, which is much less than both human and yeast DNA polymerase η. The most frequent mutation was the targeted G→T transversion (3.6%).^ The toxicity and mutagenesis of this lesion was investigated replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is carried out primarily by these DNA polymerases. In the single polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single base substitutions or deletions, though a small population of mutants carrying two point mutations at or near G[8,5-Me]T was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied based on the identity of the DNA polymerase that was knocked-out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. It was determined that pol V is required for most targeted G→T transversions, whereas pol IV is required for the targeted T deletions. The results show that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link. ^