CHARACTERIZATION OF THE METAL-ION BINDING HELIX-HAIRPIN-HELIX MOTIFS IN HUMAN DNA-POLYMERASE-BETA BY X-RAY STRUCTURAL-ANALYSIS

Citation
H. Pelletier et Mr. Sawaya, CHARACTERIZATION OF THE METAL-ION BINDING HELIX-HAIRPIN-HELIX MOTIFS IN HUMAN DNA-POLYMERASE-BETA BY X-RAY STRUCTURAL-ANALYSIS, Biochemistry, 35(39), 1996, pp. 12778-12787
Citations number
28
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
35
Issue
39
Year of publication
1996
Pages
12778 - 12787
Database
ISI
SICI code
0006-2960(1996)35:39<12778:COTMBH>2.0.ZU;2-T
Abstract
X-ray crystallographic studies have shown that DNA binding by human po lymerase beta (pol beta) occurs primarily through two structurally and sequentially homologous helix-hairpin-helix (HhH) motifs, one in the fingers subdomain and the other in the 8-kDa domain [Pelletier, H., Sa waya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996a) Biochemist ry 35, 12742-12761]. In that DNA binding by each HhH motif is facilita ted by a metal ion, we set out to determine the identity of the metal ion that most likely binds to the HhH motif in vivo. Crystal soaking e xperiments were performed on human pol P-DNA cocrystals with Mg2+, Ca2 +, Na+, and K+, the four most prevalent metal ions in the cell, and in each case a data set was collected and the resulting structure was re fined. Under the conditions tested, the HhH motifs: of pol beta have a n affinity for these biologically prevalent metal ions in the order Mg 2+ < Ca2+ ( Na+ < K+, with K+ displaying the strongest binding. Crysta ls soaked in the presence of Tl+, a commonly used spectroscopic probe for K+, were too X-ray-sensitive to establish the binding behavior of Tl+, but soaking experiments with Ba2+ and Cs+ resulted in relatively stable crystals that gave evidence of metal ion binding in both HhH mo tifs, confirming that larger monovalent and divalent metal ions are ca pable of binding to the HhH metal sites. Although Mn2+, which has been categorized as a potent polymerase mutagen, binds to the HhH motifs w ith a greater affinity than Mg2+, Mn2+ does not: bind to the HhH motif s in the presence of equimolar concentrations of Naf These results sug gest that in vivo, where Mn2+ is present only in trace amounts, Mn2+ p robably does not have a large effect on DNA binding and may instead ma nifest a mutagenic effect on pol beta primarily by distorting nucleoti de binding or by directly affecting the catalytic step [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996b) Biochem istry 35, 12762-12777]. Crystal soaking experiments with 31-kDa apoenz yme crystals show that, in the absence of DNA, the HhH motif in the fi ngers subdomain binds metal ions with either much lower occupancy or n ot at all, indicating that metal ion binding is dependent on the prese nce of the DNA substrate.