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The MER3 DNA helicase catalyzes the unwinding of holliday junctions
Takuro Nakagawa1, Richard D Kolodner
1Ludwig Institute for Cancer Research, Cancer Center and Department of Medicine, University of California San Diego School of Medicine, La Jolla, California 92093-0660, USA.
Abstract:
The MER3 protein of Saccharomyces cerevisiae is required for crossover in meiosis and has been suggested to act at the initiation of homologous pairing and the resolution of Holliday junctions. The purified MER3 protein is a DNA helicase that translocates along single-stranded DNA in the 3' to 5' direction displacing annealed DNA fragments. Here, MER3 was found to be able to unwind various double-stranded DNA (dsDNA) substrates, including a 30-bp dsDNA with a 20-nucleotide 3'-overhang, a 30-bp dsDNA with a 20-nucleotide 5'-overhang, a 50-bp dsDNA with blunt ends, and a Holliday junction with 25-bp arms, each of which had a blunt end. Efficient unwinding of the 3'-overhang substrate appeared to initiate by the binding of MER3 to the 3' single-stranded tail in a reaction that required six or more unpaired bases. Unwinding of the blunt end and 5'-overhang substrates appeared to initiate at the blunt ends of these substrates. Unwinding of the Holliday junction was more efficient than the unwinding of the blunt and 5'-overhang substrates and was influenced by Mg(2+) concentrations that cause changes in the structure of the junction. Possible roles for Holliday junction unwinding in meiotic crossover are discussed.
Insights
The MER3 protein acts as a DNA helicase, unwinding double-stranded DNA and Holliday junctions. This function is crucial for meiotic crossover in Saccharomyces cerevisiae.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MER3 protein in Saccharomyces cerevisiae is vital for meiotic crossover.
- It is hypothesized to function in homologous pairing initiation and Holliday junction resolution.
- Purified MER3 protein exhibits DNA helicase activity, moving along single-stranded DNA.
Purpose of the Study:
- To investigate the DNA unwinding capabilities of the MER3 protein.
- To characterize the substrates and conditions affecting MER3's helicase activity.
- To explore the role of MER3 in resolving DNA structures relevant to meiotic recombination.
Main Methods:
- Purification of MER3 protein from Saccharomyces cerevisiae.
- In vitro DNA helicase assays using various double-stranded DNA substrates.
- Analysis of MER3's unwinding activity on dsDNA with overhangs, blunt ends, and Holliday junctions.
- Investigation of the influence of Mg(2+) on Holliday junction unwinding.
Main Results:
- MER3 unwound various double-stranded DNA substrates, including those with 3'-overhangs, 5'-overhangs, and blunt ends.
- Unwinding initiation varied depending on the substrate: 3'-overhangs initiated at the single-stranded tail, while blunt ends and 5'-overhangs initiated at blunt ends.
- MER3 demonstrated efficient unwinding of Holliday junctions, more so than other substrates, with activity modulated by Mg(2+) concentration.
- A minimum of six unpaired bases were required for efficient initiation on 3'-overhang substrates.
Conclusions:
- MER3 protein possesses DNA helicase activity capable of unwinding diverse DNA structures, including Holliday junctions.
- The substrate specificity and initiation mechanisms provide insights into MER3's role in processing recombination intermediates.
- MER3's efficient Holliday junction unwinding activity supports its proposed function in the resolution of these structures during meiotic crossover.