A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions

Abstract

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ −/− mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.

DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass

“…The 39/100-mer p/t substrate used in this study was designed to have only As and Cs on the template strand downstream of the lesion (10), and incorporation of dATP or dCTP could occur only opposite the lesion. Using combinations of either dCTP and dGTP or dATP and dGTP, the complete TLS reaction (incorporation opposite the lesion and subsequent elongation) could be visualized as the accumulation of a +3 product, resulting in the incorporation of two dGMP opposite the two Cs at template positions +2 and +3.…”

“…We have previously shown that the efficient errorfree bypass of an 8-oxo-G lesion can be achieved via a switch from Pol δ to the more faithful Pol λ (10). To investigate whether PolDIP2 plays any role in this reaction, bypass of an 8-oxo-G lesion was tested under running start conditions, on a 39/100-mer p/t with the lesion at position +26 on the template strand and bearing only As and Cs on the template strand downstream of the lesion as previously described (10). Reactions were run in parallel under error-prone (lanes 2-7) or error-free (lanes 8-13) conditions in the presence of various combinations of Pol δ, Pol λ, and PolDIP2 (Fig.…”

“…TLS is mainly performed by the Y family Pols η, ι, and κ (15). We have shown that the X family enzyme Pol λ is also essential for the TLS across the 8-oxo-G lesion (14) both for the postreplicative MutYH-dependent base excision repair of 8-oxo-G:A mismatches (18,19) and for the bypass of 8-oxo-G lesions that can stall Pol δ during DNA replication (10). PolDIP2 has been shown to interact with Pol δ, PCNA, Rev 1, Pol ζ, and Pol η and has been proposed to act as a switch factor for Pol η (6).…”

“…Human recombinant Pol δ was expressed and purified as described in ref. 10. The full length ORF of PolDIP2 was obtained from a testis cDNA library by PCR and cloned into a modified pETM33 expression vector encoding an N-terminal GST tag.…”

Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

“…An investigation entailing six human B-, X-, and Y-family pols established that (relative to dATP) correct incorporation of dCTP opposite 8-oxoG in the presence of the proliferating cell nuclear antigen and replication protein A auxiliary proteins is 1,200-and 68-fold more efficient for pol and pol , respectively (42). An important role of pol in combination with pol ␦ for bypass of 8-oxoG was also established in vitro using mouse embryonic fibroblasts and HeLa cells (43).…”