Dataset Information

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

ABSTRACT: Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB "arm" domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

SUBMITTER: Simon MJ

PROVIDER: S-EPMC4976051 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Publications

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

Simon Michael J MJ Sokoloski Joshua E JE Hao Linxuan L Weiland Elizabeth E Lohman Timothy M TM

Journal of molecular biology 20160714 15

Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3' to 5' translocase, and RecD, a 5' to 3' translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates con ...[more]

PMID: 27422010

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Project description:UnlabelledGINS is a key component of the eukaryotic Cdc45-minichromosome maintenance (MCM)-GINS (CMG) complex, which unwinds duplex DNA at the moving replication fork. Archaeal GINS complexes have been shown to stimulate the helicase activity of their cognate MCM mainly by elevating its ATPase activity. Here, we report that GINS from the thermoacidophilic crenarchaeon Sulfolobus solfataricus (SsoGINS) is capable of DNA binding and binds preferentially to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA). Notably, SsoGINS binds more strongly to dsDNA with a 5' ssDNA tail than to dsDNA with a 3' tail and more strongly to an ssDNA fragment blocked at the 3' end than to one at the 5' end with a biotin-streptavidin (SA) complex, suggesting the ability of the protein complex to slide in a 5'-to-3' direction along ssDNA. DNA-bound SsoGINS enhances DNA binding by SsoMCM. Furthermore, SsoGINS increases the helicase activity of SsoMCM. However, the ATPase activity of SsoMCM is not affected by SsoGINS. Our results suggest that SsoGINS facilitates processive DNA unwinding by SsoMCM by enhancing the binding of the helicase to DNA. We propose that SsoGINS stabilizes the interaction of SsoMCM with the replication fork and moves along with the helicase as the fork progresses.ImportanceGINS is a key component of the eukaryotic Cdc45-MCM-GINS complex, a molecular motor that drives the unwinding of DNA in front of the replication fork. Archaea also encode GINS, which interacts with MCM, the helicase. But how archaeal GINS serves its role remains to be understood. In this study, we show that GINS from the hyperthermophilic archaeon Sulfolobus solfataricus is able to bind to DNA and slide along ssDNA in a 5'-to-3' direction. Furthermore, Sulfolobus GINS enhances DNA binding by MCM, which slides along ssDNA in a 3'-to-5' direction. Taken together, these results suggest that Sulfolobus GINS may stabilize the interaction of MCM with the moving replication fork, facilitating processive DNA unwinding.

Dataset Information

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

Publications

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation.

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