title: Saw1 localizes to repair sites but is not required for recruitment of Rad10 to repair intermediates bearing short non-homologous 3' flaps during single-strand annealing in S. cerevisiae.
abstract: SAW1 is required for efficient removal by the Rad1-Rad10 nuclease of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break (DSB) repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing. Saw1 was shown in vitro to bind flaps with high affinity, but displayed diminished affinity when flaps were short (<30 deoxynucleotides [nt]), consistent with it not being required for short flap cleavage. Accordingly, this study, using in vivo fluorescence microscopy showed that SAW1 was not required for recruitment of Rad10-YFP to DNA DSBs during their repair by SSA when the flaps were ~10 nt. In contrast, recruitment of Rad10-YFP to DSBs when flaps were ~500 nt did require SAW1 in G1 phase of cell cycle. Interestingly, we observed a substantial increase in colocalization of Saw1-CFP and Rad10-YFP at DSBs when short flaps were formed during repair, especially in G1, indicating significant recruitment of Saw1 despite there being no requirement for Saw1 to recruit Rad10. Saw1-CFP was seldom observed at DSBs without Rad10-YFP. Together, these results support a model in which Saw1 and Rad1-Rad10 are recruited as a complex to short and long flaps in all phases of cell cycle, but that Saw1 is only required for recruitment of Rad1-Rad10 to DSBs when long flaps are formed in G1.
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA.
authors.forename: Melina
authors.initials: M
authors.lastname: Mardirosian
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA.
authors.forename: Linette
authors.initials: L
authors.lastname: Nalbandyan
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA.
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Miller
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA.
authors.forename: Claire
authors.initials: C
authors.lastname: Phan
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Kelson
authors.affiliation: Department of Chemistry and Biochemistry, California State University Northridge, 18111 Nordhoff St, Northridge, CA, 91330-8262, USA. paula.fischhaber@csun.edu.
authors.forename: Paula L
authors.identifier: http://orcid.org/0000-0001-7939-5188
authors.initials: PL
authors.lastname: Fischhaber
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C579349:Saw1 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases
keywords: Double-strand break repair; Rad10; Saw1; Single-strand annealing