Chromosome integrity at a double-strand break requires exonuclease 1 and MRX

Abstract

The continuity of duplex DNA is generally considered a prerequisite for chromosome continuity. However, as previously shown in yeast as well as human cells, the introduction of a double-strand break (DSB) does not generate a chromosome break (CRB) in yeast or human cells. The transition from DSB to CRB was found to be under limited control by the tethering function of the RAD50/ MRE11/XRS2 (MRX) complex. Using a system for differential fluorescent marking of both sides of an endonuclease-induced DSB in single cells, we found that nearly all DSBs are converted to CRBs in cells lacking both exonuclease 1 (EXO1) activity and MRX complex. Thus, it appears that some feature of exonuclease processing or resection at a DSB is critical for maintaining broken chromosome ends in close proximity. In addition, we discovered a thermal sensitive (cold) component to CRB formation in an MRX mutant that has implications for chromosome end mobility and/or end-processing.

Coincident Resection at Both Ends of Random, γ–Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease

“…γ-radiation induced DSBs with dirty ends are tethered by MRX complex, which is consistent with MRX preventing a chromosome break at a clean I- Sce I cut site [20] and [21] as well as in vitro results [24]. In this model Sae2, MRX, as well as the Mre11 nuclease provide for coordinated resection.…”

“…Interestingly, the absence of MRX, Mre11 nuclease, or Sae2 can lead to gross genomic changes including BIR at a unique DSB [17]–[19]. Previously, MRX complex was found to be required in vivo to efficiently hold DSB ends together based on single molecule analysis of each end of I- Sce I-induced chromosome breaks using two different color probes close to either DSB end [20], [21] or using single color probes at positions distant from an HO-induced DSB [22]. The specific impact of the Sae2 and Mre11 nuclease mutants on coincident resection is best explained by defects in initiation of resection at dirty γ–DSBs rather than a defect in tethering since neither Sae2 nor Mre11 nuclease is required to prevent chromosome breaks [20], [21].…”

“…Previously, we and others showed that MRX is required in vivo to hold DSB ends together, based on single molecule analysis of each end of I- Sce I-induced chromosome breaks using different fluorescent probes near the DSB [20], [21] or a common probe distant from the two sides of a DSB [22]. Tethering of defined DSBs by MR (Mre11/Rad50) complex, which has been described in crystal structures [23] and is observed in vitro [24], suggests that this complex could facilitate a “co-processing” mechanism of coordinated end-processing that would protect the genome from 1-end resection events.…”

Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

“…Consistent with a role in limiting DSB persistence, genetic deficiencies in the MRE11-RAD50-NBS1 complex have been shown to cause persistent coding DSB ends during repair of inverted V(D)J recombination substrates (6). Regarding end tethering, the MRE11-RAD50-NBS1 complex promotes in vitro DNA tethering and EJ via DNA ligase III (15, 18 -20), and the MRE11-RAD50-XRS2 complex is important in Saccharomyces cerevisiae for maintaining spatial continuity of a broken chromosome and for suppressing chromosomal translocations (63,64). In summary, we suggest that the functions of DNAPKcs kinase activity and RAD50 in limiting the persistence of DSBs and/or promoting correct end tethering could contribute to their roles in limiting incorrect end use during EJ of multiple DSBs.…”

The Sister Chromatid Cohesion Pathway Suppresses Multiple Chromosome Gain and Chromosome Amplification

“…However, measuring chromosome loss does not provide a complete view of chromatid malsegregation or aneuploidy tolerance. For example, inability to repair double-strand breaks (DSBs) may cause loss of a chromosome that is not due to a defect in chromatid transmission as evident by the number of DNA repair strains that exhibit increased chromosome loss (Yuen et al 2007) and especially proteins involved in recombination (Nakai et al 2011;Song and Petes 2012). Unlike chromosome gain, loss of chromosomes cannot be measured in haploid cells that contain natural 1n complement of chromosomes, obscuring the ability to study ploidy-dependent effects on chromatid segregation.…”