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The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

ABSTRACT: The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

SUBMITTER: Rossi MJ

PROVIDER: S-EPMC3080997 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

Rossi Matthew J MJ Mazina Olga M OM Bugreev Dmitry V DV Mazin Alexander V AV

Proceedings of the National Academy of Sciences of the United States of America 20110404 16

The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do ...[more]

PMID: 21464277

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Project description:Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

Dataset Information

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

Publications

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

Similar Datasets

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