Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells.
Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants.
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Lisa
L
Rank
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Research Training Group 1331, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Sebastian
S
Veith
Bioimaging Center, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Konstanz Research School Chemical Biology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Eva C
EC
Gwosch
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Janine
J
Demgenski
Bioimaging Center, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Konstanz Research School Chemical Biology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Magdalena
M
Ganz
Department of Human Genetics, Radboud University Medical Center Nijmegen, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.|Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherland.
Marjolijn C
MC
Jongmans
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Christopher
C
Vogel
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Konstanz Research School Chemical Biology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Arthur
A
Fischbach
FlowKon FACS Facility, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Stefanie
S
Buerger
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Konstanz Research School Chemical Biology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Jan M F
JM
Fischer
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Konstanz Research School Chemical Biology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Tabea
T
Zubel
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Anna
A
Stier
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Christina
C
Renner
Center of Applied Photonics, Department of Physics, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Michael
M
Schmalz
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Ecotoxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Sascha
S
Beneke
FlowKon FACS Facility, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.|Immunology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Marcus
M
Groettrup
Department of Human Genetics, Radboud University Medical Center Nijmegen, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
Roland P
RP
Kuiper
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Alexander
A
Bürkle
Bioimaging Center, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
Elisa
E
Ferrando-May
Molecular Toxicology Group, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany aswin.mangerich@uni-konstanz.de.
Aswin
A
Mangerich
D000818:Animals; D002460:Cell Line; D004249:DNA Damage; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D009243:NAD / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D000069896:Transcription Activator-Like Effector Nucleases / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D009243:NAD; D000071137:Poly (ADP-Ribose) Polymerase-1; D000069896:Transcription Activator-Like Effector Nucleases
Mech Ageing Dev. 2010 May;131(5):366-9
20399804
Sci Signal. 2011 Apr 05;4(167):ra20
21467298
PLoS One. 2014 May 22;9(5):e98022
24853559
Mol Cell. 2015 Dec 3;60(5):742-54
26626479
Nat Genet. 2011 Jun 26;43(8):729-31
21706002
Cancer Res. 2012 Nov 1;72 (21):5588-99
23118055
Mol Aspects Med. 2013 Dec;34(6):1217-56
23370117
J Biol Chem. 1999 Jun 18;274(25):17860-8
10364231
J Biol Chem. 2008 Jan 11;283(2):1197-208
18025084
Genes Dev. 1995 Mar 1;9(5):509-20
7698643
J Biol Chem. 1995 Feb 17;270(7):3247-54
7852410
ACS Chem Biol. 2013 Jul 19;8(7):1567-75
23631432
Mutat Res. 2000 Jun 30;460(1):1-15
10856830
Mol Cell. 2015 Dec 3;60(5):755-68
26626480
Curr Opin Struct Biol. 2013 Feb;23(1):134-43
23333033
Cancer Res. 2004 Sep 1;64(17):6344-8
15342424
Mol Aspects Med. 2013 Dec;34(6):1138-52
23454615
Curr Opin Cell Biol. 2005 Aug;17(4):435-41
15978795
Nucleic Acids Res. 2007;35(21):e143
17991682
Nucleic Acids Res. 2007;35(22):7665-75
17982172
Nat Commun. 2015 Aug 19;6:8088
26286827
J Biol Chem. 2008 Aug 8;283(32):21873-80
18539602
Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):1658-63
23319653
Mol Cell. 2007 Jan 26;25(2):297-308
17244536
Exp Cell Res. 2014 Nov 15;329(1):18-25
25017100
PLoS One. 2014 Jan 28;9(1):e87057
24489833
Nucleic Acids Res. 2008 Dec;36(22):6959-76
18981049
G3 (Bethesda). 2013 Aug 07;3(8):1213-24
23550136
Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2301-4
10051636
EMBO J. 2003 May 1;22(9):2255-63
12727891
Nature. 2005 Apr 14;434(7035):913-7
15829966
Br J Pharmacol. 2014 Apr;171(8):2000-16
24684389
Arch Biochem Biophys. 1976 Aug;175(2):627-34
8713
J Biophotonics. 2008 Mar;1(1):53-61
19343635
Mol Aspects Med. 2013 Dec;34(6):1066-87
23268355
Mol Cell. 2015 Jun 18;58(6):947-58
26091343
J Biol Chem. 2012 Feb 3;287(6):4198-210
22158865
Nat Genet. 2002 Aug;31(4):379-84
12118251
Eur J Biochem. 1982 Oct;127(3):579-85
6293817
Nat Genet. 2010 Dec;42(12):1109-12
21076407
Mol Cell. 2013 Oct 24;52(2):272-85
24055347
Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10209-14
24987120
J Biol Chem. 2011 Mar 4;286(9):7149-60
21183686
Hum Mol Genet. 2006 Jun 1;15(11):1816-25
16632486
Oxid Med Cell Longev. 2012;2012:321653
23050038
Exp Gerontol. 2000 Oct;35(8):989-1002
11121685
Science. 2002 Jul 26;297(5581):606-9
12065746
J Biol Chem. 2008 Feb 15;283(7):4105-14
18055453
Nat Commun. 2016 Aug 08;7:12425
27498558
Biochem Biophys Res Commun. 2006 Mar 31;342(1):336-41
16480959
Science. 2002 Jul 12;297(5579):259-63
12114629
FASEB J. 2009 Nov;23(11):3978-89
19622798
Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3765-70
17360427
Mol Cell. 2016 May 5;62(3):432-42
27067600
Biochem Biophys Res Commun. 1995 Jul 17;212(2):317-25
7626044
Cell Rep. 2014 Sep 25;8(6):1819-31
25220464
J Proteome Res. 2013 Apr 5;12 (4):1868-80
23438649
Science. 2012 May 11;336(6082):728-32
22582261
Nat Rev Clin Oncol. 2015 Jan;12(1):27-41
25286972
J Mol Med (Berl). 2000;78(8):431-40
11097112
Cell Rep. 2014 Sep 25;8(6):1808-18
25199834
Biochemistry. 1997 Oct 7;36(40):12147-54
9315851
Nature. 2013 Aug 8;500(7461):207-11
23925245
J Biol Chem. 1999 Nov 5;274(45):32122-6
10542247
Nucleic Acids Res. 2012 Nov;40(21):10780-94
22977173
Br J Cancer. 2009 Jul 21;101(2):256-62
19568233
J Biol Chem. 1993 Oct 25;268(30):22575-80
8226768
Nat Struct Mol Biol. 2012 Jun 10;19(7):685-92
22683995
J Biol Chem. 2000 Dec 29;275(52):40974-80
11016934
DNA Repair (Amst). 2015 Aug;32:10-6
25963443
Nat Struct Mol Biol. 2013 Mar;20(3):347-54
23396353
Biochem Biophys Res Commun. 2007 Mar 2;354(1):122-6
17214964
J Biol Chem. 2005 Dec 9;280(49):40450-64
16204234
Biochim Biophys Acta. 1991 Jun 24;1078(2):179-86
1648406
ACS Chem Biol. 2013 Jan 18;8(1):179-88
23082994
Nature. 2005 Apr 14;434(7035):917-21
15829967
Nat Methods. 2013 Oct;10 (10 ):981-4
23955771
DNA Repair (Amst). 2015 Jun;30:68-79
25800440
Genes (Basel). 2012 Dec 06;3(4):779-805
24705085
Nucleic Acids Res. 2010 Jan;38(3):e14
19906733
Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7303-7
9207086
Annu Rev Biochem. 2015;84:227-63
25747399
Nucleic Acids Res. 2008 Aug;36(13):4454-64
18603595
Cell. 2006 Apr 21;125(2):371-83
16630823
Proc Natl Acad Sci U S A. 2006 May 2;103(18):7136-41
16627622