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

Abstract

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 lambda and eta. 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 lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, 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.

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

“…The incorporation of 2'-deoxyribonucleotides opposite 8-OH-Gua by human DNA pol η was analyzed in vitro. Human DNA pol η incorporates dATP and dCTP opposite the lesion with comparable efficiencies, but proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) enhance the correct 8-OH-Gua bypass [25][26][27][28][29][30]. The mammalian replicative DNA pols α and δ are error-prone against 8-OH-Gua [27,28,[42][43][44][45][46][47].…”

“…It has been shown that TLS DNA pols bypassed 8-OH-Gua in vitro [25][26][27][28][29][30][31][32][33][34]. Thus, to understand the mutagenesis and carcinogenesis processes by 8-OH-Gua, it is important to examine the contributions of the polymerases to the induction and suppression of the G:CT:A mutations caused by this lesion in living mammalian cells.…”

“…DNA pol λ was suggested to be involved in the bypass of 8-OH-Gua in the gap-filling reactions [28,50]. Taken together with the increased mutation by 8-OH-Gua:C and the decreased mutation by 8-OH-Gua:A in the cells with knocked-down pol η, this DNA pol would bypass 8-OH-Gua during DNA replication, with a higher dCTP/dATP incorporation ratio than those of the replicative DNA pols.…”

DNA polymerases in adaptive immunity

“…Because of their relaxed catalytic sites that allow them to accommodate distorted DNA structures, TLS polymerases are highly error-prone when copying normal undamaged DNA. Although studies of TLS have been largely focused on polymerases of the Y family (together with Polζ), other enzymes, such as Polµ, Polλ and Polθ, have shown robust translesion activity in vitro [49][50][51] .…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…Together with cofactors replication protein A and proliferating cell nuclear antigen, Pol k is the mammalian polymerase most able to correctly incorporate dC opposite 8-oxoG [Maga et al, 2007]. It is also cell-cycle regulated; phosphorylation by S-Cdk (Cdk2 and cyclin A) protects Pol k from ubiquitin-dependent proteolysis and stimulates expression during S and G2 phases.…”