|
When crystals of human DNA polymerase beta (pol beta) complexed with DNA
[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 transfer to the primer 3'-OH takes
place directly in the crystals, even though the DNA is blunt-ended at the active
site. Under similar crystal-soaking conditions, there is no evidence for a
reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent
metal ion utilized by most polymerases in vivo. These results suggest that one
way Mn2+ may manifest its mutagenic effect on polymerases is by promoting
greater reactivity than Mg2+ at the catalytic site, thereby allowing the
nucleotidyl transfer reaction to take place with little or no regard to
instructions from a template. Non-template-directed nucleotidyl transfer is also
observed when pol beta-DNA cocrystals are soaked in the presence of dATP and
Zn2+, but the reaction products differ in that the sugar moiety of the
incorporated nucleotide appears distorted or otherwise cleaved, in agreement
with reports that Zn2+ may act as a polymerase inhibitor rather than as a
mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436].
Although no reaction is observed when crystals 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 metal ions tested, with the exception of Mg2+, promote a change in the
side-chain position of aspartic acid 192, which is one of three highly conserved
active-site carboxylate residues. Soaking experiments with nucleotides other
than dATP (namely, dCTP, dGTP, 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, suggesting a possible mechanism for nucleotide
selectivity whereby triphosphate-sugar binding precedes a check for correct base
pairing with the template.
|
