Topoisomerase II-mediated DNA damage is differently repaired during the cell cycle by non-homologous end joining and homologous recombination.
Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2alpha was largely responsible for the induction of gammaH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells.
de Campos-Nebel
Marcelo
M
Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires, Argentina. mnebel@hematologia.anm.edu.ar
Larripa
Irene
I
González-Cid
Marcela
M
D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; C448305:RAD51C protein, human; D004250:DNA Topoisomerases, Type II
J Biol Chem. 2008 Jan 4;283(1):1-5
17999957
Mol Cell Biol. 2006 Nov;26(21):7929-41
16923961
Cancer Res. 2001 Jun 1;61(11):4550-5
11389089
J Biol Chem. 2008 Aug 29;283(35):23711-20
18596031
DNA Cell Biol. 2005 Jun;24(6):388-93
15941391
EMBO J. 1998 Sep 15;17(18):5497-508
9736627
BMC Pharmacol. 2004 Dec 02;4:31
15575955
Mol Cell Biol. 2005 Dec;25(24):10842-52
16314509
DNA Repair (Amst). 2008 Mar 1;7(3):452-63
18206427
Mol Cancer Ther. 2006 Jun;5(6):1405-14
16818498
DNA Repair (Amst). 2006 May 10;5(5):575-90
16567133
J Biol Chem. 2001 Nov 2;276(44):40652-8
11546768
Bioessays. 1998 Mar;20(3):215-26
9631649
Mol Cell Biol. 2003 Aug;23(16):5706-15
12897142
Cell. 2006 Jan 27;124(2):287-99
16439204
Electrophoresis. 1999 Jul;20(10):2133-8
10451126
DNA Repair (Amst). 2006 Sep 8;5(9-10):1075-81
16798112
Am J Hum Genet. 1997 Oct;61(4):795-800
9382087
Cell Cycle. 2008 Sep 15;7(18):2902-6
18769152
J Cell Biol. 2007 Apr 23;177(2):219-29
17438073
DNA Repair (Amst). 2006 Sep 8;5(9-10):1093-108
16857431
Biochem Biophys Res Commun. 2004 Jun 11;318(4):856-61
15147950
DNA Repair (Amst). 2007 Jul 1;6(7):923-35
17363343
Nat Genet. 2001 Mar;27(3):247-54
11242102
J Cell Sci. 2004 Mar 1;117(Pt 7):1047-54
14996935
Anal Biochem. 1976 May 7;72:248-54
942051
Cancer Lett. 2003 Apr 10;193(1):1-9
12691817
Nat Rev Mol Cell Biol. 2002 Jun;3(6):430-40
12042765
J Biol Chem. 1998 Dec 11;273(50):33660-6
9837951
Cell. 1988 Oct 7;55(1):7-16
3167977
Mol Cell Biol. 1997 Oct;17(10):6087-96
9315668
Mol Cell Biol. 1999 Jul;19(7):5166-9
10373565
Nucleic Acids Res. 2009 Feb;37(3):738-48
19042970
Cell. 1992 Sep 18;70(6):983-91
1356077
Bioessays. 1997 Feb;19(2):97-9
9046238
J Biol Chem. 1994 Jan 14;269(2):1173-6
8288578
Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9053-8
18574145
Clin Chim Acta. 2007 Apr;379(1-2):1-13
17306783
Cancer Res. 2007 Aug 1;67(15):7078-81
17671173
J Biol Chem. 2003 Sep 19;278(38):35897-902
12842886
J Cell Biol. 1999 Jan 11;144(1):11-20
9885240
J Mol Biol. 2003 Feb 7;326(1):93-103
12547193
Cancer Res. 1990 Sep 15;50(18):5778-83
2168280
Apoptosis. 2010 Feb;15(2):162-72
20041303
J Biol Chem. 1996 Nov 15;271(46):29238-44
8910583
Genes Dev. 2001 Dec 15;15(24):3308-18
11751636
Annu Rev Biochem. 1996;65:635-92
8811192
Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):11014-9
17578914
Cytometry A. 2004 Apr;58(2):99-110
15057963
J Cell Sci. 2002 Jan 1;115(Pt 1):153-64
11801733
J Biol Chem. 2005 Apr 15;280(15):14709-15
15677476
Cancer Res. 2003 Mar 15;63(6):1377-81
12649202
Br J Cancer. 1997;75(9):1340-6
9155056
Cell. 2006 Jan 27;124(2):301-13
16439205
Nucleic Acids Res. 1994 Jun 25;22(12):2274-81
8036155
Genes Dev. 2002 Sep 15;16(18):2333-8
12231622
Science. 2006 Jun 23;312(5781):1798-802
16794079