A STRUCTURAL BASIS FOR METAL-ION MUTAGENICITY AND NUCLEOTIDE SELECTIVITY IN HUMAN DNA-POLYMERASE-BETA

When crystals of human DNA polymerase beta (pol beta) complexed with D NA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transf er to the primer 3'-OH takes place directly in the crystals, even thou gh the DNA is blunt-ended at the active site. Under similar crystal-so aking conditions, there is no evidence for a reaction when Mn2+ is rep laced by Mg2+ which is thought to be the divalent metal ion utilized b y most polymerases in vivo. These results suggest that one way Mn2+ ma y manifest. its mutagenic effect on polymerases is by promoting greate r reactivity than Mg2+ at the catalytic site, thereby allowing the nuc leotidyl transfer reaction to take place with Little or no regard to i nstructions from a template. Non-template-directed nucleotidyl transfe r is also observed when pol beta-DNA cocrystals are soaked in the pres ence of dATP and Zn2+, but the reaction products differ in that the su gar moiety of the incorporated nucleotide appears distorted or otherwi se cleaved, in agreement with reports that Zn2+ may act as a polymeras e inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1 976) Science 194, 1434-1436]. Although no reaction is observed when cr ystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, Or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate; moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all met al ions tested, with the exception of Mg2+, promote a change in die si de-chain position of aspartic acid 192, which is one of three highly c onserved active-site carboxylate residues. Soaking experiments with nu cleotides other than dATP (namely, dCTP, dCTP, dTTP, ATP, ddATP, ddCTP , AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, sug gesting a possible mechanism for nucleotide selectivity whereby tripho sphate-sugar binding precedes a check for correct base pairing with th e template.