Metal-induced DNA translocation leads to DNA polymerase conformational activation
| Autor: | William A. Beard, Thomas W. Kirby, Samuel H. Wilson, David D. Shock, Geoffrey A. Mueller, Eugene F. DeRose, Nisha A. Cavanaugh, Robert E. London |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Hot Temperature
DNA polymerase Stereochemistry Base pair Cations Divalent Protein Conformation DNA polymerase beta Genome Integrity Repair and Replication 010402 general chemistry 01 natural sciences DNA polymerase delta 03 medical and health sciences chemistry.chemical_compound Genetics Magnesium Nuclear Magnetic Resonance Biomolecular Polymerase DNA Polymerase beta 030304 developmental biology 0303 health sciences DNA clamp biology DNA replication Biological Transport DNA 0104 chemical sciences Enzyme Activation Zinc chemistry Biochemistry Amino Acid Substitution biology.protein Primer (molecular biology) |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | Binding of the catalytic divalent ion to the ternary DNA polymerase β/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg(2+) and Zn(2+) on the conformational activation process based on NMR measurements of [methyl-(13)C]methionine DNA polymerase β. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|