Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

Chung, Woo Hyun; Zhu, Zhu; Papusha, Alma; Malkova, Anna; Ira, Grzegorz

doi:10.1371/journal.pgen.1000948

Cited by 156 publications

(196 citation statements)

References 56 publications

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Section: Discussionmentioning

confidence: 99%

“…Previously we demonstrated that BIR is strongly stimulated in cells under replication stress, and that many of these BIR events are initiated by breaks at FS2 . BIR events can initiate centromereproximal to the original break location due to extensive 59 to 39 end resection at DNA breaks to expose ssDNA (Chung et al 2010;Symington 2016).Both the extensive lengths and high abundance of bidirectional noncrossover tracts we observed can be explained if these tracts result not from canonical DSBR but from dBIR. The dBIR mechanism is also a better fit for our experimental system in which replication stress is likely to result in replication fork 124 S. A. Chumki et al…”

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confidence: 86%

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Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

Chumki¹,

Dunn²,

Coates³

et al. 2016

Genetics

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Replication stress causes breaks at chromosomal locations called common fragile sites. Deletions causing loss of heterozygosity (LOH) in human tumors are strongly correlated with common fragile sites, but the role of gene conversion in LOH at fragile sites in tumors is less well studied. Here, we investigated gene conversion stimulated by instability at fragile site FS2 in the yeast Saccharomyces cerevisiae. In our screening system, mitotic LOH events near FS2 are identified by production of red/white sectored colonies. We analyzed single nucleotide polymorphisms between homologs to determine the cause and extent of LOH. Instability at FS2 increases gene conversion 48-to 62-fold, and conversions unassociated with crossover represent 6-7% of LOH events. Gene conversion can result from repair of mismatches in heteroduplex DNA during synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR), and from break-induced replication (BIR) that switches templates [double BIR (dBIR)]. It has been proposed that SDSA and DSBR typically result in shorter gene-conversion tracts than dBIR. In cells under replication stress, we found that bidirectional tracts at FS2 have a median length of 40.8 kb and a wide distribution of lengths; most of these tracts are not crossover-associated. Tracts that begin at the fragile site FS2 and extend only distally are significantly shorter. The high abundance and long length of noncrossover, bidirectional gene-conversion tracts suggests that dBIR is a prominent mechanism for repair of lesions at FS2, thus this mechanism is likely to be a driver of common fragile site-stimulated LOH in human tumors.KEYWORDS loss of heterozygosity; gene conversion; BIR; fragile site; homologous recombination D NA integrity is frequently challenged by chemical, environmental, and physical agents, as well as by endogenous factors. Common fragile sites are one source of endogenous damage, causing DNA instability and breaks under conditions of replication stress when DNA replication is partially inhibited (Durkin and Glover 2007;Sarni and Kerem 2016). Replication stress has typically been induced experimentally by treating cells with the polymerase inhibitor aphidicolin (Glover et al. 1984) or by genetically lowering the levels of DNA polymerases Lemoine et al. 2008). In vivo, replication stress has been observed early in the process of tumor development as a result of alteration of replication fork progression and nucleotide deficiency caused by activation of oncogenes (Di Micco et al. 2006;Bester et al. 2011). In cancer cells, human common fragile sites are hotspots for chromosomal deletions, amplifications, and rearrangements (Burrow et al. 2009;Drusco et al. 2011; OzeriGalai et al. 2011 OzeriGalai et al. , 2012, and a large-scale screen of deletions in 746 cancer cell lines reported that many areas of unexplained deletions are in fragile sites (Bignell et al. 2010).The alterations at human common fragile sites in cancer cells are proposed to result from the processes of DNA repair at...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 86%

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

Chumki¹,

Dunn²,

Coates³

et al. 2016

Genetics

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“…Subsequent work revealed that these assays largely overestimated the length of DNA that is resected in vivo under normal conditions when repair is possible. In mitotic cells, it has been determined that ϳ2,000 -4,000 nt are resected in allelic recombination and ϳ3,000 -6,000 nt are resected in ectopic recombination (38). In meiotic cells, where the long-range resection is largely dependent on Exo1, the resection tracks are even shorter (ϳ800 nt) (39).…”

Section: Rad51-dependent Pathwaymentioning

confidence: 99%

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

Ćejka

2015

Journal of Biological Chemistry

186

199

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The repair of DNA double-strand breaks by homologous recombination commences by nucleolytic degradation of the 5-terminated strand of the DNA break. This leads to the formation of 3-tailed DNA, which serves as a substrate for the strand exchange protein Rad51. The nucleoprotein filament then invades homologous DNA to drive template-directed repair. In this review, I discuss mainly the mechanisms of DNA end resection in Saccharomyces cerevisiae, which includes short-range resection by Mre11-Rad50-Xrs2 and Sae2, as well as processive long-range resection by Sgs1-Dna2 or Exo1 pathways. Resection mechanisms are highly conserved between yeast and humans, and analogous machineries are found in prokaryotes as well. Homologous recombination (HR)2 plays a central role in the repair of DNA double-strand breaks (DSBs) (1). In vegetative cells, recombination restores broken DNA to preserve genome integrity. In meiosis, HR promotes proper chromosome segregation and exchange of genetic information between maternal and paternal genomes, and thus contributes to the generation of genetic diversity. Recombination is initiated upon the formation of ssDNA overhangs through a process termed DNA end resection. The nucleolytic processing of broken DNA ends is essential for all recombination mechanisms (Fig. 1). Resection of DSBs commits their repair to HR as it prevents ligation by the potentially more mutagenic non-homologous end-joining (NHEJ) pathway (2-4). Resected DNA is first coated by the ssDNA-binding protein replication protein A (RPA). In most cases, RPA is subsequently replaced with the strand exchange protein Rad51, forming a nucleoprotein filament capable of invading homologous DNA. Repair can then proceed via either of two main recombination pathways, synthesis-dependent strand annealing or the canonical pathway that involves the formation of a double Holliday junction (Fig. 1). Single-strand annealing (SSA) is instead a Rad51-independent pathway that requires extensive resection of DNA between two repetitive sequences ( Fig. 1). DNA End Resection: When and What to ResectDSBs can form accidentally in any phase of the cell cycle upon exposure to ionizing radiation or chemicals or as a result of abortive processing of nucleic acids. Most DSBs, however, occur in S-phase when a DNA replication fork runs into a nick. Furthermore, DSBs are sometimes introduced "intentionally," such as in the prophase of the first meiotic division or during anticancer therapy regimens based on DNA-damaging agents (5). Depending on the cellular context, cells must first "decide" whether or not to resect the breaks (3, 4, 6, 7). DNA end resection commits the repair to HR and prevents NHEJ; therefore, it would be detrimental to resect DSBs in the G 1 phase of the cell cycle when no sister chromatid DNA is available as a template for repair. Cells have thus developed regulatory control mechanisms that activate resection only during the S or G 2 phases of the cell cycle, which will be introduced below (4, 6 -8).Another critical parameter is the pola...

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“…In yeast, Pif1 helicase is suggested to be involved in mitochondrial DNA recombination, repair, and replication (6,11). Some of its nuclear functions include Okazaki fragment processing (12,13), inhibition of rDNA replication (14), unwinding of G-quadruplex structures (15)(16)(17)(18), telomere regulation (19 -21), and repair of double-stranded DNA breaks (22)(23)(24)(25). S. cerevisiae Pif1 belongs to the superfamily (SF) 2 1B class of helicases.…”

mentioning

confidence: 99%

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

Chib

Byrd

Raney

2016

Journal of Biological Chemistry

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Saccharomyces cerevisiae Pif1, an SF1B helicase, has been implicated in both mitochondrial and nuclear functions. Here we have characterized the preference of Pif1 for RNA:DNA heteroduplexes in vitro by investigating several kinetic parameters associated with unwinding. We show that the preferential unwinding of RNA:DNA hybrids is due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts. This higher processivity of Pif1 is attributed to slower dissociation from RNA:DNA hybrids. Biologically, this preferential role of the helicase may contribute to its functions at both telomeric and nontelomeric sites.

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Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting

Cited by 156 publications

References 56 publications

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

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