ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery.
Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM-dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C-terminal S/TQ residues. Suppression of ATR-mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co-localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM-unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.
Ammazzalorso
Francesca
F
Department of Environment and Primary Prevention, Section of Experimental and Computational Carcinogenesis and Section of Molecular Epidemiology, Istituto Superiore di Sanità, Rome, Italy.
Pirzio
Livia Maria
LM
Bignami
Margherita
M
Franchitto
Annapaola
A
Pichierri
Pietro
P
D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D000071657:Werner Syndrome Helicase
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase
Hum Genet. 1999 Jan;104(1):10-4
10071186
J Biol Chem. 2004 May 7;279(19):20067-75
14982920
J Cell Biol. 2008 Oct 20;183(2):241-52
18852298
Oncogene. 2003 Mar 13;22(10):1491-500
12629512
EMBO J. 1998 Jan 2;17(1):159-69
9427750
Science. 2008 Mar 7;319(5868):1352-5
18323444
Mol Biol Cell. 2001 Aug;12(8):2412-21
11514625
Mol Cell Biol. 2005 Oct;25(20):8925-37
16199871
J Cell Biol. 2008 Jan 28;180(2):305-14
18209099
Nat Rev Mol Cell Biol. 2008 Aug;9(8):616-27
18594563
J Biol Chem. 2002 Jun 28;277(26):23236-45
11956187
Carcinogenesis. 2008 Jan;29(1):52-61
17984114
Science. 2007 May 25;316(5828):1160-6
17525332
Oncogene. 2000 Dec 7;19(52):5955-63
11146546
Cell. 2002 Dec 13;111(6):779-89
12526805
EMBO Rep. 2000 Jul;1(1):80-4
11256630
J Biol Chem. 1999 Dec 31;274(53):37538-43
10608806
J Cell Sci. 2006 Dec 15;119(Pt 24):5137-46
17118963
Mech Ageing Dev. 2003 Feb;124(2):167-74
12633936
Cell. 2001 Feb 9;104(3):397-408
11239397
Curr Opin Cell Biol. 2009 Apr;21(2):237-44
19230642
Hum Mol Genet. 2005 Oct 15;14 Spec No. 2:R197-205
16244318
Biochemistry. 2006 Nov 28;45(47):13939-46
17115688
Genes Dev. 2000 Jun 15;14(12):1448-59
10859164
J Biol Chem. 2003 Dec 26;278(52):52997-3006
14534320
J Biol Chem. 2009 Feb 27;284(9):5994-6003
19049966
DNA Repair (Amst). 2006 Sep 8;5(9-10):1126-35
16807141
J Mol Biol. 2001 Apr 13;307(5):1235-45
11292338
Cancer Res. 1991 Sep 1;51(17):4671-6
1873812
Mol Cell Biol. 2005 Jan;25(2):563-74
15632059
Trends Biochem Sci. 2008 Dec;33(12):609-20
18926708
J Biol Chem. 2003 Sep 26;278(39):37139-45
12847089
J Biol Chem. 2002 Aug 23;277(34):30515-23
12034743
Hum Genet. 2008 Nov;124(4):369-77
18810497
J Biol Chem. 2004 Aug 13;279(33):34603-13
15187093
Cell Cycle. 2004 Oct;3(10):1331-9
15467456
Mutat Res. 2003 Apr 9;525(1-2):85-92
12650908
Cell Cycle. 2008 Mar 15;7(6):796-807
18250621
DNA Repair (Amst). 2008 Nov 1;7(11):1776-86
18722555
Nat Cell Biol. 2005 Feb;7(2):195-201
15665856
FASEB J. 2001 May;15(7):1224-6
11344095
EMBO J. 2000 Feb 1;19(3):463-71
10654944
Ital J Biochem. 2007 Jun;56(2):130-40
17722654
Mol Cell Biol. 2004 Feb;24(3):1279-91
14729972
Nat Struct Mol Biol. 2007 Nov;14(11):1096-104
17934473
Mol Cell. 2010 Feb 26;37(4):492-502
20188668
EMBO J. 2006 Apr 19;25(8):1764-74
16601701