Replication fork regression in vitro by the Werner syndrome protein (WRN): holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity.
The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ helicase family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regression capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP-dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11-13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the established cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks.
Machwe
Amrita
A
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA.
Xiao
Liren
L
Lloyd
Robert G
RG
Bolt
Edward
E
Orren
David K
DK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D045566:DNA, Cruciform / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D053484:RecQ Helicases / Q000378:metabolism* / Q000502:physiology; D000071657:Werner Syndrome Helicase
D045566:DNA, Cruciform; D004247:DNA; D005090:Exodeoxyribonucleases; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase
Nat Genet. 2004 Aug;36(8):877-82
15235603
Mol Cell Biol. 2004 Oct;24(19):8437-46
15367665
EMBO Rep. 2004 Oct;5(10):983-8
15375374
Hum Genet. 1988 Oct;80(2):135-9
2459043
Mech Ageing Dev. 2007 Mar;128(3):259-66
17224176
BMC Mol Biol. 2006;7:1
16412221
Nature. 2003 Dec 18;426(6968):870-4
14685245
J Biol Chem. 2004 Apr 30;279(18):18099-102
15023996
EMBO J. 2004 Jul 21;23(14):2882-91
15241474
Proc Natl Acad Sci U S A. 1989 Aug;86(15):5893-7
2762303
Exp Cell Res. 1992 Oct;202(2):267-73
1327851
Cell. 1995 Nov 17;83(4):655-66
7585968
Cell. 1996 Feb 23;84(4):497-500
8598035
Science. 1996 Apr 12;272(5259):258-62
8602509
J Biol Chem. 1997 Jun 6;272(23):14873-82
9169457
Nat Genet. 1997 Sep;17(1):100-3
9288107
Mech Ageing Dev. 1997 Dec;98(3):239-54
9352493
J Biol Chem. 1997 Dec 5;272(49):30611-4
9388193
Hum Genet. 1997 Dec;101(2):121-5
9402954
Nat Genet. 1998 Oct;20(2):114-6
9771700
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13097-102
9789047
Bioessays. 1998 Dec;20(12):985-91
10048298
Hum Genet. 1999 Jan;104(1):10-4
10071186
J Biol Chem. 1999 Apr 30;274(18):12488-98
10212225
Nat Genet. 1999 May;22(1):82-4
10319867
Trends Biochem Sci. 1999 Jul;24(7):271-5
10390616
Nucleic Acids Res. 1999 Sep 1;27(17):3557-66
10446247
Cancer Lett. 2005 Mar 10;219(2):125-35
15723711
Nat Genet. 2000 Jan;24(1):16-7
10615119
Trends Biochem Sci. 2000 Apr;25(4):156-65
10754547
Trends Biochem Sci. 2000 Apr;25(4):173-8
10754549
Trends Genet. 2000 May;16(5):213-20
10782115
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6504-8
10823897
Nucleic Acids Res. 2000 Jun 15;28(12):2396-405
10871373
Nature. 2000 Nov 9;408(6809):263-6
11089984
Science. 2001 Feb 2;291(5505):839-40
11225633
EMBO Rep. 2000 Jul;1(1):80-4
11256630
FASEB J. 2001 May;15(7):1224-6
11344095
Genes Cells. 2001 May;6(5):421-30
11380620
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8196-202
11459953
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8227-34
11459957
Mol Biol Cell. 2001 Aug;12(8):2412-21
11514625
J Biol Chem. 2002 Feb 8;277(6):4492-504
11717307
J Biol Chem. 2002 Jun 28;277(26):23236-45
11956187
Mol Cell Biol. 2002 Oct;22(20):6971-8
12242278
Nat Rev Mol Cell Biol. 2002 Nov;3(11):859-70
12415303
Biochemistry. 2002 Nov 19;41(46):13483-8
12427008
Mutat Res. 2002 Dec 29;510(1-2):107-20
12459447
Oncogene. 2003 Mar 13;22(10):1491-500
12629512
Mech Ageing Dev. 2003 Feb;124(2):167-74
12633936
Aging Cell. 2002 Oct;1(1):30-9
12882351
Biochem J. 2003 Sep 15;374(Pt 3):577-606
12803543
Hum Mol Genet. 2003 Nov 1;12(21):2837-44
12952869
J Biol Chem. 2005 Jun 17;280(24):23397-407
15845538
Nucleic Acids Res. 2005;33(12):3932-41
16024743
J Biol Chem. 2005 Jul 29;280(30):28072-84
15899892
Mech Ageing Dev. 2005 Nov;126(11):1146-58
16087220
J Biol Chem. 2005 Oct 7;280(40):34143-51
16079128
DNA Repair (Amst). 2006 Feb 3;5(2):172-80
16214424
J Med Genet. 2006 Feb;43(2):148-52
15964893
BMC Mol Biol. 2006;7:6
16503984
Front Biosci. 2006;11:2657-71
16720342
Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9154-9
16754873
J Biol Chem. 2006 Aug 11;281(32):22839-46
16766518
Nucleic Acids Res. 2006;34(18):5217-31
17003056
Biochemistry. 2006 Nov 28;45(47):13939-46
17115688