Critical Role of Magnesium Ions in DNA Polymerase 's Closing and Active Site Assembly
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- 作者：Linjing Yang ; Karunesh Arora ; William A. Beard ; Samuel H. Wilson ; and Tamar Schlick
- 刊名：Journal of the American Chemical Society
- 出版年：2004
- 出版时间：July 14, 2004
- 年：2004
- 卷：126
- 期：27
- 页码：8441 - 8453
- 全文大小：1085K
- 年卷期：v.126,no.27(July 14, 2004)
- ISSN：1520-5126
文摘
To dissect the effects of the nucleotide-binding and catalytic metal ions on DNA polymerasemechanisms for DNA repair and synthesis, aside from the chemical reaction, we investigate their roles inthe conformational transitions between closed and open states and assembly/disassembly of the activesite of polymerase 
/DNA complexes before and after the chemical reaction of nucleotide incorporation.Using dynamics simulations, we find that closing before chemical reaction requires both divalent metalions in the active site while opening after the chemical reaction is triggered by release of the catalyticmetal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conservedcatalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalyticion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group throughthe nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg2+ andNa+ ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ionitself is not a rate-limiting conformational or overall step. However, geometric adjustments associated withfunctional ions and proper positioning in the active site, including subtle but systematic motions of proteinside chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelitymechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotideapproaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding andcatalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptionsof polymerase pathways and underscores the importance of the delicate conformational events both beforeand after the chemical reaction to polymerase efficiency and fidelity mechanisms.
/DNA complexes before and after the chemical reaction of nucleotide incorporation.Using dynamics simulations, we find that closing before chemical reaction requires both divalent metalions in the active site while opening after the chemical reaction is triggered by release of the catalyticmetal ion. The critical closing is stabilized by the interaction of the incoming nucleotide with conservedcatalytic residues (Asp190, Asp192, Asp256) and the two functional magnesium ions; without the catalyticion, other protein residues (Arg180, Arg183, Gly189) coordinate the incomer's triphosphate group throughthe nucleotide-binding ion. Because we also note microionic heterogeneity near the active site, Mg2+ andNa+ ions can diffuse into the active site relatively rapidly, we suggest that the binding of the catalytic ionitself is not a rate-limiting conformational or overall step. However, geometric adjustments associated withfunctional ions and proper positioning in the active site, including subtle but systematic motions of proteinside chains (e.g., Arg258), define slow or rate-limiting conformational steps that may guide fidelitymechanisms. These sequential rearrangements are likely sensitively affected when an incorrect nucleotideapproaches the active site. Our suggestion that subtle and slow adjustments of the nucleotide-binding andcatalytic magnesium ions help guide polymerase selection for the correct nucleotide extends descriptionsof polymerase pathways and underscores the importance of the delicate conformational events both beforeand after the chemical reaction to polymerase efficiency and fidelity mechanisms.