Insight into the catalytic mechanism of DNA polymerase beta: Structures ofintermediate complexes

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in met al ion catalysis of the chemical step, Here we report a functional intermed iate structure of a DNA polymerase with only one metal ion bound, the DNA p olymerase beta -DNA template-primer-chromium(LII).2'-deoxythymidine 5'-beta ,gamma -methylenetriphosphate [Cr(III). dTMPPCP] complex, at 2.6 Angstrom r esolution. The complex is distinct from the structures of other polymerase- DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydrox yl,group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnov er in biochemical assays of crystallized protein as well as from the determ ination that soaking Pol beta crystals with Mn(LT) ions leads to formation of the product complex, Pol beta -DNA-Cr(LII). PCP, whose structure is also reported. An important feature of both structures is that the fingers subd omain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of th e fingers subdomain is induced specifically by binding of the metal-dNTP co mplex prior to binding of the catalytic Mg2+ ion. This has led us to reeval uate our previous evidence regarding the existence of a rate-limiting confo rmational change in Pol beta 's reaction pathway. The results of stopped-fl ow studies suggest that there is no detectable rate-limiting conformational change step.