Dataset Information

Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert.

ABSTRACT: The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, although the ATPase activity has been attributed to a highly conserved AAA+ family ATPase domain. Here we show that changes to N- and C-terminal motifs in the single MCM protein from the archaeon Methanothermobacter thermautotrophicus (MthMCM) can modulate ATP hydrolysis, DNA binding, and duplex unwinding. Furthermore, these motifs appear to influence the movement of the beta-alpha-beta insert in helix-2 of the MCM ATPase domain. Removal of this motif from MthMCM increased dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for ssDNA and dsDNA. Deletion of the helix-2 insert additionally resulted in the abrogation of DNA unwinding. Our results provide significant insight into the molecular mechanisms used by the MCM helicase to both regulate and execute DNA unwinding.

SUBMITTER: Jenkinson ER

PROVIDER: S-EPMC1472493 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert.

Jenkinson Elizabeth R ER Chong James P J JP

Proceedings of the National Academy of Sciences of the United States of America 20060505 20

The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, althoug ...[more]

PMID: 16679413

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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

Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert.

Publications

Minichromosome maintenance helicase activity is controlled by N- and C-terminal motifs and requires the ATPase domain helix-2 insert.

Similar Datasets

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