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
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.