title: Resolution of single and double Holliday junction recombination intermediates by GEN1.
abstract: 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-TopoisomeraseIIIα-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'.
authors.affiliation: DNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
authors.forename: Rajvee
authors.initials: R
authors.lastname: Shah Punatar
authors.affiliation: DNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
authors.forename: Maria Jose
authors.initials: MJ
authors.lastname: Martin
authors.affiliation: DNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
authors.forename: Haley D M
authors.initials: HD
authors.lastname: Wyatt
authors.affiliation: DNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
authors.forename: Ying Wai
authors.initials: YW
authors.lastname: Chan
authors.affiliation: DNA Recombination and Repair Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom stephen.west@crick.ac.uk.
authors.forename: Stephen C
authors.identifier: 0000-0001-8848-9418
authors.initials: SC
authors.lastname: West
mesh_terms: D001483:Base Sequence; D004260:DNA Repair; D045566:DNA, Cruciform / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000378:metabolism*; D059765:Homologous Recombination; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases; C533748:GEN1 protein, human; D045565:Holliday Junction Resolvases
keywords: RecA; RuvC; recombination; repair; resolvase