The Saccharomyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions
Lepakshi Ranjha1, Roopesh Anand, Petr Cejka
1From the Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Abstract:
MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX2EX4E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.
Insights
MutLγ, a protein complex crucial for meiosis, acts as a nuclease. This study shows MutLγ (Mlh1-Mlh3) binds DNA, particularly Holliday junctions, revealing its role in generating genetic crossovers.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MutLγ, a heterodimer of Mlh1 and Mlh3, is essential for meiotic homologous recombination.
- Its meiotic function depends on a nuclease motif, suggesting a role in processing recombination intermediates.
- Mechanistic understanding of MutLγ has been limited by the lack of purified recombinant protein.
Purpose of the Study:
- To express and purify the yeast Mlh1-Mlh3 heterodimer.
- To investigate the biochemical properties and DNA binding preferences of MutLγ.
- To determine if Holliday junction binding is conserved in human MLH1-MLH3.
Main Methods:
- Expression and purification of recombinant yeast Mlh1-Mlh3 complex.
- Biochemical assays to determine nuclease activity.
- DNA binding studies using various DNA substrates, including Holliday junctions.
- Expression and purification of human MLH1-MLH3 complex for comparative analysis.
Main Results:
- Recombinant MutLγ was purified to near homogeneity.
- MutLγ exhibits nuclease activity, nicking double-stranded DNA.
- MutLγ displays high-affinity DNA binding with a preference for Holliday junctions.
- This preferential binding to Holliday junctions is conserved in the human MLH1-MLH3 complex.
- MutLγ preferentially binds the open, unstacked form of Holliday junctions.
Conclusions:
- MutLγ functions as a DNA nuclease involved in meiotic recombination.
- The specific recognition and binding of Holliday junctions represent a novel function for eukaryotic MutL homologues.
- MutLγ's preference for open Holliday junctions supports its role in processing these structures to generate meiotic crossovers.
Related Concept Videos
02:31Homologous Recombination
02:31Homologous Recombination
01:20Mismatch Repair
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
01:36Mismatch Repair
01:36Mismatch Repair
02:08Gene Conversion