Dataset Information

The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain.

ABSTRACT: The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase-nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3' ? 5' polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences.

SUBMITTER: Yeeles JT

PROVIDER: S-EPMC3064778 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain.

Yeeles Joseph T P JT Gwynn Emma J EJ Webb Martin R MR Dillingham Mark S MS

Nucleic acids research 20101110 6

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase-nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. ...[more]

PMID: 21071401

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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

The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain.

Publications

The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain.

Similar Datasets

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