Molecular Basis of Selectivity of Nucleoside Triphosphate Incorporation Opposite O6-Benzylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4

Abstract

Previous work has shown thatAlkyl and aryl modifications are some of the most extensively studied forms of DNA damage (1), with O 6 -alkylation providing one of the more mutagenic examples of this type of lesion (2, 3). Alkylating agents have been used in warfare (i.e. mustard gas), and they also continue to be used in chemotherapeutic regimes (4 -6). Endogenous sources of DNA alkylation can arise through nonenzymatic methylation by S-adenosylmethionine, although O 6 -alkylation is presumed to occur to only trace extents (7). Cellular repair of O 6 -alkylation can result from the activity of O 6 -alkylG DNA-alkyltransferases as well as mismatch repair pathways (8 -10). If O 6 -alkylation (or almost any type of DNA damage for that matter) is encountered by the replisome, then replication can be impeded or stopped completely. The geometric constraints placed upon the active site of "high fidelity" polymerases often translate into an inability to effectively bypass certain types of DNA damage (11). The Y-family DNA polymerases, on the other hand, appear to be well adapted toward translesion synthesis across many types of modified DNA.Many studies have focused on DNA polymerase bypass of O 6 -alkylG 2 lesions, most concerning bypass of the mutagenic O 6 -MeG lesion (12-17). A recent study from our own group showed that the human replicative pol ␦ bypass of O 6 -alkylG (including MeG, BzG, and PobG) was inhibited in very much the same manner as several human Y-family polymerases (including pol , , and ) (18). In general, relatively little preference was observed in steady-state reactions for either C or T insertion opposite O 6 -alkylG with any of the polymerases tested, consistent with what has been reported with other polymerases. One notable difference was observed between pol ␦ * This work was supported by National Institutes of Health Grants R01 ES010375 (to F. P. G.), F32 CA119776 (to R. L. E.), P30 ES000267 (to F. P. G. and M. E.), and P01 ES05355 (to M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2jef, 2jeg, 2jei, and 2jej) have PobG,butylguanine; pol, (DNA) polymerase; pol T7 Ϫ , bacteriophage pol T7 exonuclease-deficient; RT, reverse transcriptase; UDG, uracil DNA glycosylase; r.m.s., root mean square. The generic term "alkyl" is used to include both alkyl and aralkyl (Bz) groups for convenience.

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

“…Similarly in the case of S. solfataricus Dpo4, hydrogen bonding is more important than base pair geometry for nucleotide selectivity opposite O 6 -methylG. The crystal structures of Dpo4 with O 6 -methylG and O 6 -benzylG have shown that a wobble base pair forms when dCTP pairs opposite the lesion (13,16). However, unlike pol , Dpo4 utilizes wobble base pairing to preferentially incorporate dCTP opposite O 6 -methylG, thus maintaining high fidelity during lesion bypass (13).…”

“…The crystal structure of the Y-family DNA polymerase Sulfolobus solfataricus Dpo4 revealed wobble base pairing of cytosine opposite O 6 -methylG (13). The NMR results and crystal structures demonstrate that the O 6 -methylG:T base pair forms a pseudo Watson-Crick pair, with a single hydrogen bond formed between the O2 atom of T and the N2 atom of O 6 -methylG (11,13,14,16). The shape of the Watson-Crick-like O 6 -methylG:T base pair fits in the active site of the DNA polymerase without distorting the DNA, thereby contributing to the incorporation of dTTP opposite the lesion (17).…”

Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene

“…Dpo4 (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)). In fact, the asymmetric loss of efficiency observed with Klenow fragment (large for d(DFT)TP:A and small for dATP:DFT) could be due to different influences of hydrogen bonds from polymerase residues to template and dNTP bases.…”

Binary complex crystal structure of DNA polymerase β reveals multiple conformations of the templating 8-oxoguanine lesion

“…In general, however, once the template 8-oxoG:A base pair is incorporated into DNA and is at the template-primer terminus during DNA synthesis, it is well-extended by DNA polymerases facilitating G to T mutagenesis. The structural basis for DNA polymerase preferences in mutagenic bypass of the 8-oxoG lesion is emerging across the DNA polymerase families (8)(9)(10)(11)(12) and is the subject of the present report.…”