NMR study of the effect of Zn on conformational activation of rat DNA polymerase β

Abstract

The DNA repair enzyme DNA Polymerase β (Polβ) fills gapped DNA using a two‐metal ion (usually Mg2+) mechanism. Crystal structures show that the transition from an “open” binary complex (Polβ·gapped DNA) to a “closed” ternary complex (Polβ·gapped DNA·dNTP) activates formation of the active site geometry involved in Mg2+ coordination. Other divalent metals can also function in place of magnesium, and some can cause a decrease in fidelity. We have investigated the effect of Zn by using [methyl‐13C] methionine‐labeled Polβ in NMR experiments to study the conformational changes undergone by Polβ in solution during its catalytic cycle The methionine probes are located in the lyase domain (M18), and in different parts of the polymerase domain, i.e. the catalytic subdomain (M155, M158, M191, and M236); and the nascent base‐pair binding subdomain (M282). The transitions that correspond to the “open” to “closed” structures (with Mg2+ present) can be monitored by the chemical shifts of the methionine methyl groups. In particular, the large chemical shift change and broadening of the M282 resonance detects the rearrangement of helix N (in the nascent base‐pair binding subdomain) induced by the formation of the activated ternary complex. In this work we show that these same responses can be achieved for a binary complex in the presence of Zn2+ ions. (Supported by the Intramural Research Program of the National Institutes of Health)