Structural and Functional Elucidation of the Mechanism Promoting Error-prone Synthesis by Human DNA Polymerase κ Opposite the 7,8-Dihydro-8-oxo-2′-deoxyguanosine Adduct

Abstract

Human polymerase kappa (hPol ) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished "bursts" for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase .DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery, and various mechanisms exist to either remove the resulting lesions or bypass them in a more or less mutation-prone fashion (1). Error-prone polymerases are central to trans-lesion synthesis across sites of damaged DNA (2, 3). Four so-called Y-class DNA polymerases have been identified in humans, Pol , 4 Pol , Pol , and Rev1, which exhibit different activities and abilities to replicate past a flurry of individual lesions (4, 5). Homologs have also been identified and characterized in other organisms, notably DinB (Pol IV) in Escherichia coli (6 -8), Dbh in Sulfolobus acidocaldarius (9, 10), and Dpo4 in Sulfolobus solfataricus (11,12). A decade of investigations directed at the structural and functional properties of bypass polymerases have significantly improved our understanding of this class of enzymes (5, 13). A unique feature of Y-class polymerases, compared with the common right-handed arrangement of palm, thumb, and finger subdomains of high fidelity (i.e. A-class) DNA polymerases (14), is a "little finger" or "PAD" (palm-associated domain) subdomain that plays a crucial role in lesion bypass (12,(15)(16)(17)(18)(19)(20)(21). In addition to the little finger subdomain at the C-terminal end of the catalytic core, both Rev1 and Pol exhibit an N-terminal extension that is absent in other translesion polym...

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

“…The LC-MS/ MS-based analyses of the full-length extension products of in vitro primer bypass past 8-oxoG carried out for all three polymerases (Table 5) (this work and Refs. 30,32) are also supportive of the similar behaviors of hpol and Dpo4, i.e. nearly errorfree bypass of 8-oxoG and relatively error-prone bypass by hpol FIGURE 6.…”

“…These observations support the general view that Dpo4 displays a behavior that is more similar to that of hpol but is clearly distinct from its genetic homolog, hpol . We reported earlier that hpol bypasses 8-oxoG in an error-prone fashion (32). For example, the k p values of insertions of dATP and dCTP opposite the adduct were 8.2 and 0.4 s Ϫ1 , respectively, and only the former process was consistent with a burst phase.…”

“…However, we observed that the pol homolog Dpo4 from Sulfolobus solfataricus bypasses 8-oxoG with high fidelity (19:1 ratio of dCTP:dATP incorporation opposite 8-oxoG) and efficiency (pre-steady-state kinetics revealed faster rates of dCTP incorporation opposite 8-oxoG than G) (30) that could be attributed to a significant extent to Arg-332 from the LF domain (31). By comparison, we established that human pol (hpol ) bypasses 8-oxoG in an error-prone fashion, with dATP rather than dCTP preferentially incorporated opposite the lesion (32). Its proclivity to insert A opposite 8-oxoG resembles the action of replicative polymerases (33,34).…”

Structural basis for the binding and incorporation of nucleotide analogs with L -stereochemistry by human DNA polymerase λ

“…Crystals were obtained using the hanging drop vapor diffusion method in which each Polλ ternary complex mixture was equilibrated against a reservoir buffer composed of 0.1 M sodium cacodylate (pH 6.5), 0.2 M calcium acetate, and 4% (wt/vol) PEG8000 (36). Notably, noncatalytic Ca(II), rather than catalytic Mg(II), was used here, as Ca (II) has been used regularly to trap incoming nucleotides in preinsertion ternary complexes with other DNA polymerases (37)(38)(39)(40)(41). Crystals were harvested and placed in cryosolutions in four different steps of increasing PEG [4-18% (wt/vol)] and ethylene glycol [12.5% (vol/vol)] concentrations before they were flash frozen in liquid nitrogen (36).…”

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

“…Similarly, TLS efficiency and specificity have been examined after swapping the LF domain (32), the finger domain (33), or the linker between the thumb and LF among Y-family members (34). In terms of lesion specificity, amino acid substitution of residue Arg332 in the LF of Dpo4 (35) and Leu508 in the LF of hPolκ (36) have been shown to alter the efficiency of 8-oxo-G bypass. Studies of BPDE-dG bypass are limited.…”