Functional Interplay of the Mre11 Nuclease and Ku in the Response to Replication-Associated DNA Damage

Abstract

The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11⌬ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2⌬ mutants in an Exo1-dependent manner. CPT-induced G 2 /M arrest, ␥-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease-and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer.

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

“…It is likely that these yku mutants exhibit reduced affinity for DNA ends, as do those described by Petrini and colleagues 40 . If the basis of suppression of sae2D sgs1Dmutant phenotype by yku mutations is to restore end resection by allowing Exo1 access to DSBs arising during DNA replication 36,37,40,41 , then the rescue should be dependent on HR. We found that deleting RAD52 reduced the size of the sae2D sgs1D yKu70D spore colonies ( Supplementary Fig.…”

“…Because deletion of yKU was shown to rescue sae2D and sae2D ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6004 sgs1D defects and to cause an acute shortening of telomeres [36][37][38] , we sequenced YKU70 and yKU80 in five independent clones showing pattern I (Fig. 2).…”

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

“…In the case of KU70 (orf19.1135 ), since it is a key player in NHEJ (Chico et al 2011), one of the pathways for DSB repair (Critchlow and Jackson 1998;Daley et al 2005), it would not be surprising that Caku70 null strains are sensitive to high MMS concentrations. Indeed, S. cerevisiae ku70 mutants are sensitive to MMS (Foster et al 2011); besides, in this background, telomeres become shorter and cause thermosensitivity at 37° (Barnes and Rio 1997). Since C. albicans is intrinsically more thermotolerant than S. cerevisiae, analysis of the thermosensitivity of Caku70 null strains required incubation at higher temperatures (42-43°).…”

Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins

“…What is the biological significance underlying the specific binding of ScMre11 to intermediates of recombination and repair? A large body of evidence supports the view that the Mre11 complex plays a key role in the process of DNA replication (65)(66)(67)(68)(69)(70); however, the mechanism is less well characterized. The observation that Mre11 exhibits striking selectivity toward replication fork and flayed duplex is consistent with its demonstrated role during "normal" replication or under conditions of replicative stress (70).…”

“…Moreover, recent studies have shown that Mre11 complex, specifically its nuclease activity, plays an essential role in processing of the stalled replication forks, thereby helping to maintain genome integrity during replication stress (65)(66)(67)(68)(69)(70). To understand the mechanism of processing of stalled replication forks and other intermediates by MRX complex better, we considered whether ScMre11 could unwind replication and recombination intermediates, such as the Holliday junction and flayed duplex substrates.…”