The DNA-binding domain of human PARP-1 interacts with DNA single-strand breaks as a monomer through its second zinc finger.
Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1+F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1+F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair.
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Sebastian
S
Eustermann
Hortense
H
Videler
Ji-Chun
JC
Yang
Paul T
PT
Cole
Dominika
D
Gruszka
Dmitry
D
Veprintsev
David
D
Neuhaus
D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016335:Zinc Fingers
D002843:Chromatin; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases
Biochem J. 1999 Sep 1;342 ( Pt 2):249-68
10455009
EMBO J. 2009 Sep 2;28(17):2601-15
19629035
Acta Crystallogr D Biol Crystallogr. 1995 Mar 1;51(Pt 2):127-35
15299312
Eur J Biochem. 1987 Dec 1;169(2):365-72
3121314
Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22090-5
21127267
J Am Chem Soc. 2001 Sep 5;123(35):8634-5
11525687
FEBS Lett. 1996 Dec 16;399(3):313-6
8985170
J Biol Chem. 1993 Oct 25;268(30):22575-80
8226768
Biochemistry. 2008 May 27;47(21):5804-13
18452307
J Cell Sci. 2001 Oct;114(Pt 20):3771-8
11707529
Biophys J. 2010 Oct 20;99(8):2636-46
20959105
Nat Cell Biol. 2007 Oct;9(10):1175-83
17891139
Oncogene. 2007 Sep 13;26(42):6244-52
17404575
Nat Rev Mol Cell Biol. 2006 Jul;7(7):517-28
16829982
J Biol Chem. 1999 Jul 30;274(31):21679-87
10419478
Biophys J. 2000 Mar;78(3):1606-19
10692345
J Mol Biol. 2004 Aug 27;341(5):1145-59
15321712
J Mol Biol. 1994 Jan 21;235(3):1062-71
8289308
Proc Natl Acad Sci U S A. 1988 Jan;85(1):99-102
3124104
Nucleic Acids Res. 2009 Jun;37(11):3723-38
19372272
J Biomol NMR. 2001 Nov;21(3):275-80
11775744
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3423-7
6808510
J Mol Biol. 2010 Feb 5;395(5):983-94
19962992
Biochemistry. 2003 Oct 28;42(42):12409-17
14567702
Nature. 1994 Sep 22;371(6495):346-7
8090205
Bioessays. 2004 Aug;26(8):882-93
15273990
J Biol Chem. 1983 Dec 25;258(24):15371-5
6317693
J Biol Chem. 1988 May 25;263(15):6993-9
3130376
J Biomol NMR. 2001 Jul;20(3):223-31
11519746
J Biomol NMR. 2002 Apr;22(4):317-31
12018480
Biochemistry. 2004 Jan 13;43(1):217-23
14705948
J Biol Chem. 2002 Jun 28;277(26):23675-83
11948185
Genes Dev. 2005 Sep 1;19(17):1951-67
16140981
Nucleic Acids Res. 2004 Jul 19;32(13):e107
15263063
J Biol Chem. 2008 Feb 15;283(7):4105-14
18055453
J Biol Chem. 1990 Dec 15;265(35):21907-13
2123876
Cell. 1995 Jun 2;81(5):801-9
7774019
Cancer Res. 1993 Sep 1;53(17):3976-85
8358726
Biochemistry. 1989 Mar 7;28(5):2034-40
2541771
J Mol Biol. 2005 May 6;348(3):609-15
15826658
J Mol Biol. 1989 Nov 5;210(1):229-33
2511329
EMBO J. 1993 May;12(5):2109-17
8491199
FEBS J. 2008 Mar;275(5):883-93
18215166
J Biol Chem. 2005 Apr 29;280(17):17076-83
15737996
Cell. 2004 Dec 17;119(6):803-14
15607977
J Mol Biol. 2004 Aug 13;341(3):723-38
15288782
J Biol Chem. 2002 Jun 21;277(25):23028-36
11948190
Exp Hematol. 2003 Jun;31(6):446-54
12829019
J Biol Chem. 1980 Nov 10;255(21):10502-8
6253477
DNA Repair (Amst). 2007 Apr 1;6(4):443-53
17118715
Nature. 1980 Feb 7;283(5747):593-6
6243744
Biophys Chem. 2004 Mar 1;108(1-3):165-85
15043928
Proc Natl Acad Sci U S A. 1990 Apr;87(8):2990-4
2109322
Mol Cell. 2009 Nov 13;36(3):365-78
19917246
J Magn Reson. 2003 Jan;160(1):65-73
12565051
J Biol Chem. 2010 Jun 11;285(24):18877-87
20388712
Nature. 1995 Jul 6;376(6535):37-43
7596430
J Biomol NMR. 2002 Nov;24(3):171-89
12522306
Trends Biochem Sci. 1994 Apr;19(4):172-6
8016868
DNA Repair (Amst). 2008 Jul 1;7(7):1077-86
18468963
Nat Struct Mol Biol. 2010 Feb;17(2):241-3
20098424
Curr Biol. 1998 Jan 15;8(2):R49-51
9427640
J Magn Reson. 1998 Apr;131(2):373-8
9571116