Title

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

RIS ID

94081

Publication Details

Geertsema, H. J., Kulczyk, A. W., Richardson, C. C. & van Oijen, A. M. (2014). Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery. Proceedings of the National Academy of Sciences of USA, 111 (11), 4073-4078.

Abstract

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.

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Link to publisher version (DOI)

http://dx.doi.org/10.1073/pnas.1402010111