Resolving Holliday Junctions with Escherichia coli UvrD Helicase

• Published in: The Journal of biological chemistry Vol. 287; no. 11; pp. 8126 - 8134
• Main Authors: Carter, Annamarie S., Tahmaseb, Kambiz, Compton, Sarah A., Matson, Steven W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.03.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Deoxyribonuclease I - chemistry; DNA and Chromosomes; DNA Footprinting - methods; DNA Helicase; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Recombination; DNA Repair; DNA Replication; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Cruciform - chemistry; DNA, Cruciform - genetics; DNA, Cruciform - metabolism; DNA-binding Protein; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Holliday Junction; MutL Proteins; MutS DNA Mismatch-Binding Protein - chemistry; MutS DNA Mismatch-Binding Protein - genetics; MutS DNA Mismatch-Binding Protein - metabolism; Nucleic Acid Heteroduplexes - chemistry; Nucleic Acid Heteroduplexes - genetics; Nucleic Acid Heteroduplexes - metabolism; Recombination; UvrD Helicase; UvrD Helicase; Recombination; DNA Repair; DNA Replication; DNA-binding Protein; DNA Recombination; DNA Helicase; Holliday Junction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.314047

The Escherichia coli UvrD helicase is known to function in the mismatch repair and nucleotide excision repair pathways and has also been suggested to have roles in recombination and replication restart. The primary intermediate DNA structure in these two processes is the Holliday junction. UvrD has been shown to unwind a variety of substrates including partial duplex DNA, nicked DNA, forked DNA structures, blunt duplex DNA and RNA-DNA hybrids. Here, we demonstrate that UvrD also catalyzes the robust unwinding of Holliday junction substrates. To characterize this unwinding reaction we have employed steady-state helicase assays, pre-steady-state rapid quench helicase assays, DNaseI footprinting, and electron microscopy. We conclude that UvrD binds initially to the junction compared with binding one of the blunt ends of the four-way junction to initiate unwinding and resolves the synthetic substrate into two double-stranded fork structures. We suggest that UvrD, along with its mismatch repair partners, MutS and MutL, may utilize its ability to unwind Holliday junctions directly in the prevention of homeologous recombination. UvrD may also be involved in the resolution of stalled replication forks by unwinding the Holliday junction intermediate to allow bypass of the blockage. The ability of UvrD, a DNA helicase, to unwind a Holliday junction has not been directly tested. UvrD catalyzed robust unwinding of a Holliday junction producing a forked structure. UvrD unwinds a Holliday junction by binding to the junction and translocating along opposite arms. This result is likely to have relevance in recombination and replication.