Journal Article

Collaborative coupling between polymerase and helicase for leading-strand synthesis

Maria Manosas, Michelle M. Spiering, Fangyuan Ding, Vincent Croquette and Stephen J. Benkovic

in Nucleic Acids Research

Volume 40, issue 13, pages 6187-6198
Published in print July 2012 | ISSN: 0305-1048
Published online March 2012 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gks254

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Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions.

Journal Article.  8046 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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