Abstract
The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.
Highlights
DNA double-strand breaks (DSBs) are potentially lethal lesions if unrepaired, and their misrepair can give rise to chromosomal rearrangements, a hallmark of cancer cells [1,2]
To investigate the role of Rqh1 in genome stability, we examined the relationship between Rqh1 and other DNA recombination genes in the cellular response to DSBs
Different repair and misrepair events can be quantitated by determining genetic marker loss following HO endonuclease induction of a site-specific DSB at the MATa site inserted within a non-essential minichromosome, Ch16MGH, derived from chromosome III (Figure 1B)
Summary
DNA double-strand breaks (DSBs) are potentially lethal lesions if unrepaired, and their misrepair can give rise to chromosomal rearrangements, a hallmark of cancer cells [1,2] To maintain both viability and genome stability in response to such lesions cells have evolved two types of DSB repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Rad52Sp, together with the auxiliary heterodimers Rad55Sp–Rad57Sp or Swi5Sp–Sfr1Sp mediate the loading of the RecA homologue, Rad51Sp onto the ssDNA overhang to create a Rad nucleoprotein filament This structure facilitates a homology search and strand exchange between the broken end and the homologous sequence to form a displacement-loop (D-loop) structure [10,11,12,13]. Second-end capture and ligation can result in a double-Holliday junction structure, which can be resolved with or without crossovers, a process involving Yen, Mus81Sp–Eme1Sp, or dissolved through the activities of BLM-Top (reviewed in [14])
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