title: Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining.
abstract: Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5' resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5'-overhanging DSBs (5' DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3' DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5' DSBs were rejoined more efficiently than 3' DSBs, consistent with a robust recruitment of NHEJ proteins to 5' DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5' DSBs and facilitated protection of 3' DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5' DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3' and 5' DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5' DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5' DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Zhuobin
authors.initials: Z
authors.lastname: Liang
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Sham
authors.initials: S
authors.lastname: Sunder
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Sivakumar
authors.initials: S
authors.lastname: Nallasivam
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA wilsonte@umich.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wilson
mesh_terms: D024509:3' Flanking Region; D024506:5' Flanking Region; D019457:Chromosome Breakage; D015825:Chromosomes, Fungal / Q000737:chemistry* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D059014:High-Throughput Nucleotide Sequencing; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011003:Ploidies; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C086929:XRS2 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D019951:DNA Polymerase beta; C084892:POL4 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; D010727:Phosphoric Diester Hydrolases; C505435:Tdp1 protein, S cerevisiae