title: Genome-destabilizing effects associated with top1 loss or accumulation of top1 cleavage complexes in yeast.
abstract: Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. We investigated the effects of Top1 on genome stability in Saccharomyces cerevisiae using two different assays. First, a sectoring assay that detects loss of heterozygosity (LOH) on a specific chromosome was used to measure reciprocal crossover (RCO) rates. Features of individual RCO events were then molecularly characterized using chromosome-specific microarrays. In the second assay, cells were sub-cultured for 250 generations and LOH was examined genome-wide using microarrays. Though loss of Top1 did not destabilize single-copy genomic regions, RCO events were more complex than in a wild-type strain. In contrast to the stability of single-copy regions, sub-culturing experiments revealed that top1 mutants had greatly elevated levels of instability within the tandemly-repeated ribosomal RNA genes (in agreement with previous results). An intermediate in the enzymatic reaction catalyzed by Top1 is the covalent attachment of Top1 to the cleaved DNA. The resulting Top1 cleavage complex (Top1cc) is usually transient but can be stabilized by the drug camptothecin (CPT) or by the top1-T722A allele. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in RCOs and greatly increased instability within the rDNA and CUP1 tandem arrays. A detailed analysis of the events in strains with elevated levels of Top1cc suggests that recombinogenic DNA lesions are introduced during or after DNA synthesis. These results have important implications for understanding the effects of CPT as a chemotherapeutic agent.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.
authors.forename: Sabrina L
authors.initials: SL
authors.lastname: Andersen
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.
authors.forename: Roketa S
authors.initials: RS
authors.lastname: Sloan
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Petes
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.
authors.forename: Sue
authors.initials: S
authors.lastname: Jinks-Robertson
mesh_terms: D002166:Camptothecin / Q000494:pharmacology; D003434:Crossing Over, Genetic; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D016681:Genome, Fungal; D042822:Genomic Instability; D009154:Mutation; D012335:RNA, Ribosomal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology
chemical_list: D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin
references.citation: Genetics. 1993 Apr;133(4):799-814
references.pmid: 8385050
references.citation: Microbiol Mol Biol Rev. 1999 Jun;63(2):349-404
references.pmid: 10357855
references.citation: Stat Med. 1998 Apr 30;17(8):857-72
references.pmid: 9595616
references.citation: Cold Spring Harb Perspect Biol. 2013 Nov;5(11):a012740
references.pmid: 24097900
references.citation: Mol Cell. 1998 Sep;2(3):329-40
references.pmid: 9774971
references.citation: Cell. 1988 Nov 4;55(3):413-25
references.pmid: 2902925
references.citation: Nat Struct Mol Biol. 2012 Apr;19(4):417-23
references.pmid: 22388737
references.citation: Annu Rev Biochem. 2001;70:369-413
references.pmid: 11395412
references.citation: J Biol Chem. 1997 May 9;272(19):12801-8
references.pmid: 9139740
references.citation: Yeast. 1994 Dec;10(13):1793-808
references.pmid: 7747518
references.citation: Proc Natl Acad Sci U S A. 2011 Feb 1;108(5):1987-92
references.pmid: 21245305
references.citation: DNA Repair (Amst). 2013 Mar 1;12(3):205-11
references.pmid: 23305949
references.citation: Genetics. 1989 May;122(1):19-27
references.pmid: 2659436
references.citation: Eukaryot Cell. 2004 Feb;3(1):82-90
references.pmid: 14871939
references.citation: Genetics. 2014 Sep;198(1):181-92
references.pmid: 24990991
references.citation: Annu Rev Genet. 1999;33:261-311
references.pmid: 10690410
references.citation: Proc Natl Acad Sci U S A. 2014 May 27;111(21):E2210-8
references.pmid: 24799712
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7383-8
references.pmid: 20231456
references.citation: Cell. 1989 Feb 24;56(4):619-30
references.pmid: 2645056
references.citation: Mutat Res. 2004 Mar 22;547(1-2):123-32
references.pmid: 15013706
references.citation: Yeast. 1999 Oct;15(14):1541-53
references.pmid: 10514571
references.citation: J Biol Chem. 2002 Oct 4;277(40):37207-11
references.pmid: 12151411
references.citation: Genes Dev. 2010 Jul 15;24(14):1546-58
references.pmid: 20634320
references.citation: PLoS Genet. 2009 Mar;5(3):e1000410
references.pmid: 19282969
references.citation: PLoS Genet. 2013 Apr;9(4):e1003434
references.pmid: 23593029
references.citation: Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):692-7
references.pmid: 21177431
references.citation: Clin Cancer Res. 1995 Apr;1(4):369-76
references.pmid: 9815993
references.citation: Proc Natl Acad Sci U S A. 1977 Nov;74(11):5091-5
references.pmid: 337310
references.citation: Nucleic Acids Res. 2013 May;41(9):4913-25
references.pmid: 23535143
references.citation: ACS Chem Biol. 2013 Jan 18;8(1):82-95
references.pmid: 23259582
references.citation: Mol Cell Biol. 1986 Apr;6(4):1158-63
references.pmid: 3537699
references.citation: Cell. 2002 Jan 25;108(2):183-93
references.pmid: 11832209
references.citation: Biochim Biophys Acta. 1998 Oct 1;1400(1-3):63-81
references.pmid: 9748506
references.citation: Nucleic Acids Res. 2014;42(16):10226-34
references.pmid: 25159610
references.citation: Mol Cell. 2012 Apr 27;46(2):115-24
references.pmid: 22541554
references.citation: Curr Genet. 1983 Sep;7(5):347-55
references.pmid: 24173415
references.citation: Annu Rev Genet. 2010;44:113-39
references.pmid: 20690856
references.citation: G3 (Bethesda). 2014 Nov;4(11):2259-69
references.pmid: 25236733
references.citation: Genetics. 2012 Apr;190(4):1267-84
references.pmid: 22267500
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 22;103(34):12819-24
references.pmid: 16908833
references.citation: Genetics. 1992 Oct;132(2):387-402
references.pmid: 1427035
references.citation: Science. 1995 Jun 16;268(5217):1616-9
references.pmid: 7777859
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1482-7
references.pmid: 20080586
references.citation: Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):698-703
references.pmid: 21177427
references.citation: Genome Res. 2011 Mar;21(3):477-86
references.pmid: 21173034
references.citation: Proc Natl Acad Sci U S A. 1988 Oct;85(20):7501-5
references.pmid: 2845409
references.citation: Proc Natl Acad Sci U S A. 1984 Jan;81(2):337-41
references.pmid: 6364141
references.citation: Nat Rev Mol Cell Biol. 2002 Jun;3(6):430-40
references.pmid: 12042765
references.citation: PLoS Genet. 2013 Oct;9(10):e1003894
references.pmid: 24204306
references.citation: PLoS Genet. 2008 Jun;4(6):e1000105
references.pmid: 18584028
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12825-30
references.pmid: 17652520
references.citation: Ann N Y Acad Sci. 2000;922:65-75
references.pmid: 11193926
references.citation: Cell. 1989 Jun 16;57(6):975-85
references.pmid: 2544296
references.citation: Mutat Res. 2001 May 9;476(1-2):63-9
references.pmid: 11336984