Sulfolobus solfataricus DNA Polymerase Dpo4 Is Partially Inhibited by “Wobble” Pairing between O6-Methylguanine and Cytosine, but Accurate Bypass Is Preferred

Abstract

We examined the effect of a single O 6 -methylguanine (O 6 -MeG) template residue on catalysis by a model Y family polymerase, Dpo4 from Sulfolobus solfataricus. Mass spectral analysis of Dpo4-catalyzed extension products revealed that the enzyme accurately bypasses O 6 -MeG, with C being the major product (ϳ70%) and T or A being the minor species (ϳ20% or ϳ10%, respectively), consistent with steady-state kinetic parameters. Transient-state kinetic experiments revealed that k pol , the maximum forward rate constant describing polymerization, for dCTP incorporation opposite O 6 -MeG was ϳ6-fold slower than observed for unmodified G, and no measurable product was observed for dTTP incorporation in the pre-steady state. The lack of any structural information regarding how O 6 -MeG paired in a polymerase active site led us to perform x-ray crystallographic studies, which show that "wobble" pairing occurs between C and O 6 -MeG. A structure containing T opposite O 6 -MeG was solved, but much of the ribose and pyrimidine base density was disordered, in accordance with a much higher K m,dTTP that drives the difference in efficiency between C and T incorporation. The more stabilized C:O 6 -MeG pairing reinforces the importance of hydrogen bonding with respect to nucleotide selection within a geometrically tolerant polymerase active site.Of the myriad forms that covalent modification of DNA can take, alkylation of the purine/pyrimidine bases is one of the most extensively studied (1, 2). The term "alkylating agent" encompasses a variety of known carcinogenic chemicals ranging from the spontaneously reactive nitrogen and sulfur mustards (e.g. mechlorethamine) and N-alkyl-N-nitrosoureas to metabolically activated compounds such as cyclophosphamide and N-nitrosamines (3). Guanine is generally considered the most easily oxidized of the bases, and the N7 position is the most nucleophilic atom of guanine. One prevalent form of guanine oxidation occurs at the O6 position, with a simple and widely studied process being addition of a methyl group to form O 6 -MeG 2 (4). Methylation of the O6 atom results in alternate pairing schemes that include a O 6 -MeG:C "wobble" pairing and a pseudo-"Watson-Crick" O 6 -MeG:T pair ( Fig. 1), and the relevance of O 6 -MeG to mutagenesis is well established (5-7). Cells can repair O 6 -MeG by recognition and/or removal of the lesion through either the mismatch repair pathway or through the actions of (O 6 -alkylguanine DNA alkyltransferase) (8 -10). O 6 -MeG is even observed in the DNA of the general population, although the level measured between studies has varied (4, 11). Substantially increased levels of O 6 -MeG are found in patients treated with chemotherapeutic regimes that include methylating agents (4, 12, 13).Of the enzymes associated with what has commonly been referred to as "translesion synthesis," the Y-family DNA polymerases are thought to represent the major constituent present during post-replication repair of covalently modified DNA (14 -16). Four human Y-family polymerases ar...

Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

“…For pols and , which require Watson-Crick base pairing for efficient catalysis (50,51), dCTP is more efficiently incorporated opposite N 2 -G adducts than O 6 -G adducts, because optimal Watson-Crick hydrogen bonding is possible only with N 2 -G adducts but not with O 6 -G adducts. This view would be consistent with recent reports that the S. solfataricus Y family DNA polymerase Dpo4 forms a wobble base pair between C and O 6 -MeG (or O 6 -BzG), and thus the polymerization is inhibited (52,53 may uniquely utilize an Arg for the optimal hydrogen bonding with an incoming dCTP, as shown with yeast Rev1 (34). The Hoogsteen edge of template G forms hydrogen bonds with the G loop residues of REV1, and thus the relatively large lesion O 6 -PobG (but not O 6 -MeG or O 6 -BzG), might sterically clash with the G-loop of REV1 to interfere with catalysis (compared with N 2 -adducts, which are sterically unhindered) (34).…”

Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

“…Using this modified HPLC-ESI Ϫ -MS/MS methodology, it was possible to characterize primer extension products using only 100 pmol of modified oligonucleotide. In comparison, the previous analytical methods required 1-4 nmol of modified oligonucleotide and large amounts of recombinant polymerases (28,30,40,44,45). The observed CID spectra of the primer extension products were in good agreement with the predicted CID spectra (Figs.…”

“…In their approach, a uracil residue is introduced into the primer, and the extension products are cleaved with UDG/hot piperidine to facilitate their sequencing by tandem mass spectrometry. This methodology has been previously applied in polymerase bypass studies of several DNA lesions (28,30,40,44,45). We adopted a similar methodology to sequence the in vitro replication products of 1,N 6 -␥-HMHP-dA containing templates by HPLC-ESI-MS/MS (Figs.…”

“…5-7 and Schemes 3-5). To enhance HPLC-ESI Ϫ -MS/MS detection sensitivity, a capillary HPLC column was employed instead of the conventional 1.0 -2.0-mm inner diameter columns used previously (28,30,40,44,45). Using this modified HPLC-ESI Ϫ -MS/MS methodology, it was possible to characterize primer extension products using only 100 pmol of modified oligonucleotide.…”

Bypass of Mutagenic O6-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

“…(O 6 -pobG) and O 6 -benzylguanine (O 6 -BnG) using the archeal TLS Pol Sulfolobus solfataricus Dpo4 [20][21][22][23] and with eukaryotic TLS Pols η, ι, κ and ζ. [24][25][26] These O 6 -alkylguanine adducts are relevant in terms of endogenous methylation, tobacco nitrosamine exposure, and the model hepatocarcinogen methylbenzylnitrosamine.…”