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Spatial control of the GEN1 Holliday junction resolvase ensures genome stability.

ABSTRACT: Holliday junction (HJ) resolvases are necessary for the processing of persistent recombination intermediates before cell division. Their actions, however, need to be restricted to the late stages of the cell cycle to avoid the inappropriate cleavage of replication intermediates. Control of the yeast HJ resolvase, Yen1, involves phosphorylation changes that modulate its catalytic activity and nuclear import. Here, we show that GEN1, the human ortholog of Yen1, is regulated by a different mechanism that is independent of phosphorylation. GEN1 is controlled exclusively by nuclear exclusion, driven by a nuclear export signal (NES) that restricts GEN1 actions to mitosis when the nuclear membrane breaks down. Construction of a nuclear-localized version of GEN1 revealed that its premature actions partially suppress phenotypes associated with loss of BLM and MUS81, but cause elevated crossover formation. The spatial control of GEN1 therefore contributes to genome stability, by avoiding competition with non-crossover promoting repair pathways.

SUBMITTER: Chan YW

PROVIDER: S-EPMC4172962 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage.

Project description:Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1's catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance.

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A novel role of human holliday junction resolvase GEN1 in the maintenance of centrosome integrity.

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The human Holliday junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant.

Project description:A key step in meiotic recombination involves the nucleolytic resolution of Holliday junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Delta strains results in Holliday junction resolution and crossover formation during meiosis.

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The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability.

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Bacillus subtilis RecU Holliday-junction resolvase modulates RecA activities.

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Resolution of single and double Holliday junction recombination intermediates by GEN1.

Project description:Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIII?-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'-A/TTTG/C-3'.

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Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus.

Project description:The 2.15-A structure of Hjc, a Holliday junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday junction complex is proposed, based on the available functional and structural data.

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Crystal structure of RuvC resolvase in complex with Holliday junction substrate.

Project description:The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein-DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.

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Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2.

Project description:Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.

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Regulation of Mus81-Eme1 Holliday junction resolvase in response to DNA damage.

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| S-EPMC3978046 | biostudies-literature

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