8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

摘要

Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols—belonging to the B, Y and X classes—during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols λ and η. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol λ, and 68-fold by DNA pol η, respectively. Experiments with DNA-pol-λ-null cell extracts suggested an important role for DNA pol λ. On the other hand, DNA pol ι, together with DNA pols α, δ and β, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.