Kinetics of dCTP incorporation opposite to 7,8-dihydro-8-oxoguanine with different 5' nearest neighbors by yeast polymerase

Abstract

Translesion synthesis (TLS), an important mechanism in cells refers to bypassing the DNA damage blockage on replication fork. Yeast TLS polymerase eta (poleta) is able to bypass 7,8-dihydro-8-oxoguanine (8-oxoG) on DNA with high fidelity by incorporation of dCTP opposite 8-oxoG rather than dATP to avoid G to T transversion mutation. We have shown the 5' nearest base next to 8-oxoG affects the G to T mutation by yeast and human poleta previously. In this study, the insertion efficiency of dCTP opposite 8-oxoG in various DNA sequences was kinetically investigated using yeast poleta. Based on K(m) and V(max), we demonstrated that the insertion efficiencies were also influenced by the 5' neighboring nucleotide next to 8-oxoG. The lowest V(max)/K(m) was observed when cytosine was 5' neighbouring base to 8-oxoG, in agreement with previous results in which dCTP incorporation to 8-oxoG was lowest when cytosine is on the 5'-side next to the lesion.