Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae

Abstract

Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATRlike kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.Genome instability | Global versus local checkpoint | Replication error | Mec1 | Rad53

Novel Connections Between DNA Replication, Telomere Homeostasis, and the DNA Damage Response Revealed by a Genome-Wide Screen for TEL1/ATM Interactions in Saccharomyces cerevisiae

“…Additionally, recent studies have uncovered a TEL1-dependent role in the preservation of fork stability through the prevention of fork reversion and degradation into abnormal cruciform structures (Doksani et al 2009). Consistent with this, Kaochar et al (2010) showed that tel1-D exhibits an increased frequency of dicentric chromosomes due to the fusion of inverted repeats likely due to fork reversion. As the reason why tel1-D cells exhibit short telomeres is poorly understood, it is formally possible that a failure to preserve fork stability in telomeric regions in tel1-D cells is causative for the short telomere phenotype [telomeres are enriched for replication pause sites such as G-quadruplex structures (Ivessa et al 2002;Bochman et al 2012)].…”

Rif1 and Exo1 regulate the genomic instability following telomere losses

“…We observed an Exo1‐dependent loss of genes, as well as gene duplication events, in cells lacking telomeres. Exo1 has been previously documented to facilitate or inhibit chromosomal duplication following other type of insults, through a checkpoint‐dependent (Kaochar et al ., 2010) or homology‐directed repair (HDR) dependent mechanism (Štafa et al ., 2014). However, the palindrome formation in PAL cells is both HDR‐independent (PALs are rad52∆ ) and checkpoint‐independent (in rad9∆ and rad24∆ strains) and therefore occurs by a different mechanism.…”

Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast

“…In budding yeast, cells lacking RAD9 resect DSBs faster and more extensively (Chen et al 2012; Clerici et al 2014; Lazzaro et al 2008), and while it remains unknown how Rad9-mediated resection block is achieved, it likely involves multiple mechanisms. One mechanism apparently relies on Rad9-mediated activation of Rad53, which promotes Rad53-dependent phosphorylation and inhibition of Exo1 (Cotta-Ramusino et al 2005; Jia et al 2004; Kaochar et al 2010; Morin et al 2008; Segurado and Diffley 2008). This Rad53-mediated inhibition of resection is possibly complemented by the ability of Rad9 to oligomerize and form a physical block surrounding the DSB.…”