Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast
2009; Springer Nature; Volume: 28; Issue: 21 Linguagem: Inglês
10.1038/emboj.2009.265
ISSN1460-2075
AutoresHelen Tinline-Purvis, Andrew P. Savory, Jason K. Cullen, Anoushka Davé, Jennifer B. Moss, Wendy L Bridge, Samuel Marguerat, Jürg Bähler, Jiannis Ragoussis, Richard Mott, Carol Walker, Timothy C. Humphrey,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle1 October 2009free access Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast Helen Tinline-Purvis Helen Tinline-Purvis CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Andrew P Savory Andrew P Savory CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Jason K Cullen Jason K Cullen CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Anoushka Davé Anoushka Davé CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Jennifer Moss Jennifer Moss CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Wendy L Bridge Wendy L Bridge CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Samuel Marguerat Samuel Marguerat Department of Genetics, Evolution and Environment and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Jürg Bähler Jürg Bähler Department of Genetics, Evolution and Environment and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Jiannis Ragoussis Jiannis Ragoussis The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Richard Mott Richard Mott The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Carol A Walker Carol A Walker CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Timothy C Humphrey Corresponding Author Timothy C Humphrey CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Helen Tinline-Purvis Helen Tinline-Purvis CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Andrew P Savory Andrew P Savory CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Jason K Cullen Jason K Cullen CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Anoushka Davé Anoushka Davé CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Jennifer Moss Jennifer Moss CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Wendy L Bridge Wendy L Bridge CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Samuel Marguerat Samuel Marguerat Department of Genetics, Evolution and Environment and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Jürg Bähler Jürg Bähler Department of Genetics, Evolution and Environment and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Jiannis Ragoussis Jiannis Ragoussis The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Richard Mott Richard Mott The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Carol A Walker Carol A Walker CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Timothy C Humphrey Corresponding Author Timothy C Humphrey CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK Search for more papers by this author Author Information Helen Tinline-Purvis1, Andrew P Savory1, Jason K Cullen1, Anoushka Davé1, Jennifer Moss1, Wendy L Bridge1, Samuel Marguerat2, Jürg Bähler2, Jiannis Ragoussis3, Richard Mott3, Carol A Walker1 and Timothy C Humphrey 1 1CRUK-MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, Oxfordshire, UK 2Department of Genetics, Evolution and Environment and UCL Cancer Institute, University College London, London, UK 3The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, Oxfordshire, UK *Corresponding author. Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Oxford, Oxfordshire OX3 7DQ, UK. Tel.: +44 1865 617327; Fax: +44 1865 617318; E-mail: [email protected] The EMBO Journal (2009)28:3400-3412https://doi.org/10.1038/emboj.2009.265 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double-strand break (DSB)-induced extensive LOH, and its suppression, using a non-essential minichromosome, Ch16, in fission yeast. We find extensive LOH to arise from a new break-induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1-dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break-induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome-wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair. Introduction Loss of heterozygosity (LOH), in which the remaining functional allele of a gene is lost, can lead to tumourigenesis through loss of tumour suppressor function (Knudson, 1993). LOH is detected at high frequency in both sporadic and hereditary cancers, and often extends several megabases encompassing whole chromosome arms (Lasko et al, 1991). Such extensive LOH may result from a variety of chromosomal rearrangements, including mitotic non-disjunction, truncations, interstitial deletions and translocations (Jasin, 2000). DNA double-strand breaks (DSBs) are potential initiating events leading to chromosomal aberrations (Pfeiffer et al, 2000). DSBs can result from exposure to DNA damaging agents, such as ionizing radiation, but can also arise spontaneously as a result of normal DNA metabolism (Shrivastav et al, 2008). Cells have evolved two distinct pathways to repair DSBs: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is initiated by resection of the broken ends, followed by strand invasion of a homologous template (Krogh and Symington, 2004). Resection during DSB repair requires the MRX/MRN complex (Mre11–Rad50–Xrs2 in Saccharomyces cerevisiae (Sc), Mre11–Rad50–Nbs1 in Schizosaccharomyces pombe (Sp) and Homo sapiens (Hs)) (Paques and Haber, 1999; Llorente et al, 2008), Exo1(Tsubouchi and Ogawa, 2000; Tomita et al, 2003; Llorente and Symington, 2004) and Sae2Sc/Ctp1Sp/CtIPHs, (Clerici et al, 2005; Limbo et al, 2007; Sartori et al, 2007; Huertas et al, 2008). In addition, roles for Sgs1Sc/BLMHs helicase, and Dna2Sc in resection have recently been identified (Gravel et al, 2008; Mimitou and Symington, 2008; Zhu et al, 2008). After such end processing, a Rad51 (Rhp51Sp) nucleoprotein filament is formed, which is facilitated by Rad52 (Rad22Sp) and the heterodimeric Rad55–Rad57 (Rhp55Sp-Rhp57Sp) mediator complex (Sung, 1997a, 1997b). The nucleoprotein filament facilitates homology search and strand invasion leading to the formation of a displacement (D) loop structure (Sugawara et al, 2003; Wolner et al, 2003). Current models suggest that the invaded strand is a substrate for three distinct subpathways of HR: double-strand break repair (DSBR), synthesis-dependent strand annealing (SDSA) and break-induced replication (BIR) (Paques and Haber, 1999; Llorente et al, 2008). In the classic model of HR (DSBR), DNA synthesis is associated with extension of the D-loop, thus facilitating second end capture. After branch migration and ligation, this results in the formation of a double-Holliday junction, which is subsequently resolved either with or without crossovers associated with gene conversion (Szostak et al, 1983). Although crossovers between identical sister chromatids do not compromise genome integrity, HR between homologous chromosomes results in gene conversion and can lead to extensive LOH (Jasin, 2000). During SDSA, the invading strand is expelled from the homologous template after DNA synthesis, resulting in the re-annealing of the two broken chromosome arms. As SDSA minimizes crossovers, it is postulated to occur more frequently during mitotic growth. BIR or recombination-induced replication initiates extensive replication after processing and strand invasion of a single broken end. BIR is thought to be important for repair of stalled or collapsed replication forks, and in telomere maintenance in the absence of telomerase. Both Rad51-dependent and -independent forms have been documented, and recently BIR has been shown to require both leading and lagging strand DNA polymerases (Malkova et al, 1996, 2005; Davis and Symington, 2004; Cullen et al, 2007; Lydeard et al, 2007). BIR can also cause chromosomal rearrangements and extensive LOH by long-distance replication using a homologous chromosome template, ectopic sites, and by template switching (Malkova et al, 1996; Smith et al, 2007; VanHulle et al, 2007; Llorente et al, 2008). In this study, we have investigated the mechanisms by which a site-specific DSB can give rise to extensive LOH, and how such mechanisms are suppressed, using a non-essential minichromosome in fission yeast. Unexpectedly, we find that a single DSB can lead to extensive LOH resulting from isochromosome formation, in which the broken chromosome arm is resected to the centromere and replaced by an inverted copy of the intact arm. In addition, we have identified roles for homologous recombination genes and the MRN complex in suppressing isochromosome formation. These findings together support a model in which extensive end processing arising from failed or inefficient HR repair leads to chromosomal rearrangements, and extensive LOH. Results Extensive LOH is associated with loss of the broken chromosome arm To screen for suppressors of extensive break-induced LOH, a strain carrying a non-essential minichromosome (Ch16), experimentally derived from the centromeric region of chromosome III (ChIII) (Niwa et al, 1986), was adapted such that extensive break-induced LOH, resulting from loss of the distal arm of Ch16 could be detected using a colony-sectoring assay. An HO endonuclease target site, MATa, together with an adjacent kanMX6 gene, encoding G418 resistance, was integrated into the right arm of Ch16, centromere-proximal to the ade6-M216 heteroallele and his3 marker. An arg3 marker was integrated into the left arm of the minichromosome, 300 kb away from the break site, to form Ch16-RMGAH (Figure 1A). In this context, extensive break-induced LOH resulting from the HO endonuclease expression would be expected to result in arg+ G418S ade− his− cells, which at high levels could be detected as red sectoring colonies on plates containing low levels of adenine following loss of the ade+ marker (Materials and methods). To examine break-induced LOH, HO endonuclease was expressed from a plasmid (pREP81X-HO) in the absence of thiamine, and colony phenotypes were quantified. Following DSB induction, 20% of the colonies were arg+ G418R ade+ his+, consistent with repair through end joining (NHEJ) or sister chromatid conversion (SCC); 53% of the colonies were arg+ G418S ade+ his+, consistent with repair by interchromosomal gene conversion (GC); and 16% of the colonies were arg− G418S ade− his− as a result of minichromosome loss through failed DSB repair. In addition, 10% of colonies were arg+ G418S ade− his− consistent with having undergone extensive LOH, but were not easily visualized using a colony sectoring assay (Figures 1B and 6A). This level of break-induced LOH was significantly higher than that previously observed (Cullen et al, 2007). Extensive LOH was break dependent, as loss of the markers distal to the break site was not observed after incubation for 48 h with a blank plasmid. To investigate the mechanism of break-induced LOH in Ch16-RMGAH, the chromosomes of 25 arg+ G418S ade− his− colonies were examined by pulsed field gel electrophoresis (PFGE). Although no size changes were detected in endogenous chromosomes of these colonies (Figure 1C), PFGE revealed that these colonies possessed a significantly smaller chromosomal element of 388 kb, instead of the uncut 530-kb Ch16-RMGAH (Figure 1D, lanes 2–4). Figure 1.LOH is associated with broken chromosome arm loss in wild-type background. (A) Schematic of Ch16-RMGAH. Ch16-RMGAH, ChIII, centromeric regions (ovals), complementary heteroalleles (ade6-M216 and ade6-M210; white), and the his3 marker (vertical stripes), ∼50 kb centromere-distal to ade6-M216, are as previously shown (Cullen et al, 2007). The MATa site (black) with an adjacent kanMX6 resistance marker gene (grey) was inserted into spcc23B6.06 ∼30 kb centromere-proximal to ade6-M216. The arg3 marker was inserted into spcc1795.09 on the left arm of the minichromosome. Derepression of pREP81X-HO (data not shown) generates a DSB at the MATa target site (scissors). The distance from the MATa site to the centromere is shown. In Ch16-RMHAH, kanMX6 is replaced by hph (B) Percentage DSB-induced marker loss in wild-type backgrounds using strains TH2130-3 and TH2357 (Supplementary Table 5). The levels of non-homologous end joining/sister chromatid conversion (NHEJ/SCC), gene conversion (GC), minichromosome loss (Ch16 loss), and LOH are shown. s.e.m. values are indicated. (C) PFGE analysis of chromosomal DNA from wild-type strain containing Ch16-RMGAH (TH2130; lane1), and individual wild-type arg+ G418S his−ade− strains isolated after DSB induction (lane 2–4). (D) High-resolution PFGE analysis of the strains described above. Southern blot analysis of the PFGE shown in (D) probed with arg3 (E; probe 1), spcc4b3.18 (F; probe 2) and tel1 ∼10 kb centromere-proximal to the MATa site (G; probe 3). Download figure Download PowerPoint To determine the structure of this new chromosomal element Southern blot analysis was carried out. An arg3 probe was able to anneal to both the parental Ch16-RMGAH minichromosome and to the chromosomal elements from three individually isolated arg+ G418S ade− his− colonies, consistent with the arg+ phenotype of these cells (Figure 1E, probe 1). Similarly, a chk1 probe was able to anneal to both parental minichromosomes and to the new chromosomal elements (see Figure 3B). These data indicated that the left arm of the minichromosome was maintained in the shorter chromosomal element. Numerous probes targeted to regions distal to the break site, while annealing to the parental Ch16-RMGAH minichromosome, failed to anneal to this chromosomal element in any arg+ G418S ade− his− colony (our unpublished results). Similarly, probes targeted ∼10 kb centromere-proximal to the break site (probe 2) or close to the centromere ( 1. Blue indicates signal intensity <1. (D) CGH of an isolated isochromosome against a wild-type strain without a minichromosome (TH400) showing three endogenous chromosomes. Colour coding as above. Download figure Download PowerPoint Figure 5.Analysis of the isochromosome centromere. (A) Schematic of predicted centromere structure of Cen-Ch16 based on homologous cen3, with the break point of Ch16-RMGAH and Ch16-YAMGH-derived LOH colonies, indicated by an arrow. cnt3, innermost (imr3), outer (otr3) and irc3 repeats are shown. (B) Log2 ratio of the fold coverage from Illumina GAII sequencing across isolated isochromosome and minichromosome DNA (log2 of isochromosome/minichromosome) from strains TH4313 and TH2125, respectively. Sequence data aligned to S. pombe ChIII reference sequence at positions indicated (C) PCR amplification of irc3L and irc3R using irc3-R and irc3-F primers, digested using ApoI, (D) PCR amplification of imr3–otr3 junctions using imr-out and dh primers (E) PCR amplification of imr3L–cnt3 junction using primers cnt3-L and imr3-in (F) PCR amplification of cnt3–imr3R junction using primers cnt3-R and imr3-in, separated on a 2% agarose gel and stained with EtBr. Diagnostic PCR product sizes are shown. PCR amplification was carried out as described previously (Nakamura et al, 2008). Download figure Download PowerPoint Analysis of the isochromosome centromere To investigate the centromere structure of the isochromosome further, an isochromosome and parental minichromosome were isolated and sequenced using an Illumina GAII with 36-bp short reads. Sequence analysis of the isochromosome confirmed the absence of the broken right arm and the presence of an intact left arm, although no additional ectopic sequences were identified. Moreover, no sequence junctions were identified at or near the break site, suggesting that duplication of the left arm was not associated with an ectopic invasion of non-homologous sequence. Sequence comparison between the isochromosome and the parental minichromosome revealed over 100 short deletions within the isochromosome centromere ranging from 1 to 264 bp in length. These regions do not to correspond to any obvious sequence structure (our unpublished results). The variation in depth of coverage across the isochromosome and minichromosome was compared, as this is indicative of the copy number at any particular locus. Using the Schizosaccharomyces pombe genome sequence as reference, Figure 5B shows the variation in log2 ratio of coverage. Such analysis indicated that although there was a 100–500-fold overall coverage of both the minichromosome and isochromosome, the left arm of the isochromosome had approximately twice the sequence coverage compared with that of the minichromosome, consistent with the duplication of the left arm. Analysis of the centromeric region indicated that although the otr3L repeats had been duplicated, the imr3L, cnt3 and imr3R regions corresponding to central domain had not been duplicated, suggesting that the isochromosome was functionally monocentric (Pidoux and Allshire, 2004). Furthermore, the irc3L and irc3R regions seem not to have been duplicated. This analysis also identified short repeated sequence gaps in otr3R and irc3R regions, which corresponded, in part, to the deleted regions identified from the sequence analysis, suggesting that these sequences were absent from the isochromosome, and/or that otherwise identical repeats derived from elsewhere were being aligned to this region (Figure 5B). To determine the position at which the right centromeric arm was lost and the left arm was duplicated, PCR analysis was carried out. irc3L differs from irc3R by 120 bp, which contains an ApoI site (Nakamura et al, 2008). PCR amplification and ApoI digestion revealed the presence of the irc3R sequence within the minichromos
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