Dynamics of Site Switching in DNA Polymerase

详细信息    查看全文

• 作者：Rajan Lamichhane ; Svitlana Y. Berezhna ; Joshua P. Gill ; Edwin Van der Schans ; David P. Millar
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：March 27, 2013
• 年：2013
• 卷：135
• 期：12
• 页码：4735-4742
• 全文大小：380K
• 年卷期：v.135,no.12(March 27, 2013)
• ISSN：1520-5126

文摘

DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3鈥?end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3鈥?5鈥?exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule F枚rster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.