Mec1/Tel1-dependent phosphorylation of Slx4 stimulates Rad1-Rad10-dependent cleavage of non-homologous DNA tails.
Budding yeast Slx4 interacts with the Rad1-Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single-strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3' non-homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double-strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3' NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified a specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1-Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.
Toh
Geraldine W-L
GW
MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Sugawara
Neal
N
Dong
Junchao
J
Toth
Rachel
R
Lee
Sang Eun
SE
Haber
James E
JE
Rouse
John
J
D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D004277:DNA, Single-Stranded; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D004247:DNA; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C505436:SLX4 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes
Annu Rev Biochem. 2008;77:229-57
18275380
Mol Cell Biol. 1995 Apr;15(4):2245-51
7891718
Nucleic Acids Res. 2008 Jan;36(1):1-9
17962301
Methods Mol Biol. 1999;113:403-15
10443437
Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9214-9
9256462
Mol Cell Biol. 1988 Sep;8(9):3619-26
3065620
Biochem Soc Trans. 2009 Jun;37(Pt 3):495-510
19442243
Mol Cell. 2009 Jul 10;35(1):116-27
19595721
Annu Rev Biochem. 2008;77:259-87
18518821
Mol Cell Biol. 1997 Nov;17(11):6765-71
9343441
Mol Cell. 2000 May;5(5):789-99
10882115
Biochem J. 2005 Oct 15;391(Pt 2):325-33
15975089
Cell. 2009 Jul 10;138(1):63-77
19596235
Mol Cell. 2008 May 9;30(3):325-35
18471978
Yeast. 1998 Jul;14(10):953-61
9717241
Science. 1992 Oct 16;258(5081):480-4
1411547
Mol Cell. 2002 Aug;10(2):373-85
12191482
Microbiol Mol Biol Rev. 1999 Jun;63(2):349-404
10357855
Curr Biol. 2004 Dec 14;14(23):2096-106
15589151
EMBO J. 2000 Oct 16;19(20):5552-61
11032822
Mol Cell Biol. 1992 Jul;12(7):3041-9
1620114
Cell. 2009 Jul 10;138(1):78-89
19596236
Cell. 1998 Aug 7;94(3):399-409
9708741
Genetics. 1996 Mar;142(3):693-704
8849880
Nucleic Acids Res. 2008 Dec;36(21):6720-7
18948279
Annu Rev Genet. 2004;38:233-71
15568977
J Biol Chem. 1995 Oct 20;270(42):24638-41
7559571
Mol Cell Biol. 2000 Jul;20(14):5300-9
10866686
Genetics. 2005 Feb;169(2):563-74
15489516
Mol Cell Biol. 2007 Sep;27(18):6433-45
17636031
Methods Enzymol. 2002;350:141-64
12073310
Genetics. 1996 Mar;142(3):727-36
8849883
Genetics. 2008 Aug;179(4):1807-21
18579504
Cell. 1999 Feb 5;96(3):415-24
10025407
Genetics. 1999 Apr;151(4):1409-23
10101166