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

Abstract

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...

Mitotic gene conversion can be as important as meiotic conversion in driving genetic variability in plants and other species without early germline segregation

“…This is highly similar to the previous estimate (34 CO versus 16 NCO-GC, 2.12:1) at the SD1 locus in meiosis [62]. Furthermore, 29.2% (here a total of 17 NCO-GC and 7 CO-GC, 7/(7+14) = 29.2%) of the conversion events in spontaneous mitotic recombination in our data are associated with COs, which is close to estimates of mitotic recombination events in yeast (27%, χ 2 = 0.086, P = 0.77) [27,46] or meiotic recombination at the SD1 locus in rice (33.3%, χ 2 = 0.27, P = 0.604) [62].…”

High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast

“…Previously, we showed that an inverted pair of Ty elements resulted in a spontaneous mitotic recombination hotspot (termed 'HS4') on the right arm of chromosome IV ( St Charles and Petes, 2013 ). We subsequently showed that HS4-associated conversion events were associated with very long (>25 kb) conversion tracts ( Yim et al, 2014 ); conversion tracts associated with a similar pair of inverted Ty elements on chromosome III also have very long conversion tracts (median length of 41 kb; Chumki et al, 2016 ). To monitor the activity of this hotspot in an mlh1 strain, we inserted the hphMX4 and URA3 markers centromere-proximal and centromere-distal to HS4 on one of the two homologs in the mlh1 diploid YYy311.…”

Cis- and Trans-Modifiers of Repeat Expansions: Blending Model Systems with Human Genetics

“…Realignment of broken repeat ends can occur between any two segments of the repeat, assuring that a length change will frequently occur. This can potentially happen during BIR, as mentioned above, as well as during single-strand annealing (SSA) or synthesis-dependent strand annealing (SDSA) [95–100]. That being said, sister chromatid exchange may have a stabilizing role in repeat maintenance [93,101,102].…”