Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae
Introduction
Repair of spontaneous or induced DNA double-strand breaks (DSBs) is accomplished by at least two major pathways in eukaryotic organisms, called nonhomologous end-joining (NHEJ) and homologous recombination. Repair by NHEJ is mediated in Saccharomyces cerevisiae (budding yeast) by the actions of the Rad50/Mre11/Xrs2 (Mrx) nuclease, the DNA end-binding Yku70/Yku80 heterodimer and DNA Ligase IV, composed of Lif1, Dnl4 and Nej1 [1], [2], [3]. Other proteins, including Rad27 (Fen1), Pol4, Smc cohesins and the Rsc nucleosome remodeling complex, have also been implicated in NHEJ [4], [5]. In yeast cells the SIR2, SIR3 and SIR4 genes are also required for NHEJ, though their role appears to be indirect, involving regulation of NEJ1 expression [3].
Repair of DSBs by homologous recombination requires members of the Rad52 group of DNA repair proteins, including Rad51–Rad59, the Mrx complex, the Rpa single-stranded DNA binding protein, as well as several other proteins associated with strand exchange, DNA synthesis, heteroduplex strand separation and DNA ligation [6], [7]. Recombination models have been proposed that involve initial resection of DSB ends by the Mrx nuclease to generate single-stranded 3′ overhangs, followed by homology search and strand exchange reactions mediated by complexes containing Rad51, Rad52, etc. [6], [7]. The precise role of Mrx in resection remains obscure and the complex may perform additional functions such as recruitment of the Rad51 recombinase and/or the Rsc chromatin remodeling complex [5], [6], [7]. A second nuclease, Exo1, possesses a 5′-to-3′ exonuclease activity that can also generate 3′ tails at DSB ends, though this reaction is inefficient at physiological levels of the enzyme [7], [8], [9].
Yeast rad50, mre11 and xrs2 single mutants exhibit several similar phenotypes, including: (i) defects in both NHEJ and homologous recombination assays, (ii) sensitivity to ionizing radiation and strand-breaking chemicals such as methyl methanesulfonate (MMS), bleomycin, or hydroxyurea (HU), (iii) stable but shortened telomeres, (iv) defective meiosis, (v) increased chromosome and arm loss, (vi) elevated loss-of-heterozygosity (LOH) and mutation frequencies, and (vii) defects in DNA damage-responsive cell cycle checkpoints [8], [10], [11], [12], [13], [14].
The ends of chromosomes in most eukaryotic organisms contain arrays of short, repeated DNA sequences referred to as telomeres. Stable maintenance of DNA ends in dividing cells requires telomerase, an RNA-dependent DNA polymerase complex. Yeast telomerase has at least four protein subunits, called Est1, Est2, Est3 and Cdc13, as well as a 1301 nt RNA subunit encoded by the TLC1 gene [15]. Est2 is the catalytic (polymerase) subunit and Est1 and Cdc13 are DNA binding proteins thought to mediate association of the complex with chromosome ends.
The RNA subunit of telomerase acts as a scaffold for the binding of several proteins, including Est1, Est2 and Yku70/Yku80, and for association with another TLC1 RNA molecule to form dimers in vivo [16], [17], [18], [19]. Altering cellular levels of telomerase affects cell physiology in different ways. For example, expression of TLC1 RNA at supraphysiological levels disrupts silencing of transcription at telomeres and can also suppress killing of yku70 or yku80 mutants at elevated temperatures [20], [21], [22], [23]. Modulation of TLC1 expression also affects the proportion of Est2 molecules bound to the yeast PinX1 protein (Pxr1), a possible regulator of Est2 localization in the nucleus [24]. In the current study we have investigated the mechanism by which telomerase RNA overexpression alleviates the DNA repair defects of mrx cells, but not those of other DSB repair mutants. These experiments have revealed new connections between telomerase and DNA repair and have identified a new role for the Ku complex in regulation of DSB repair by the homologous recombination pathway.
Section snippets
Strains and plasmids
Yeast strains used in the study included VL6α (MATα ura3-52 his3-Δ200 trp1-Δ63 lys2-801 ade2-101 met14) [25] and BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) [26]. Gene disruptions of yeast strains were performed as described in [8]. Derivatives of VL6α included YLKL499 (rad50Δ::hisG), YLKL500 (yku70Δ::TRP1 rad50Δ::hisG), YLKL529 (mre11Δ::G418r), YLKL544 (dnl4Δ::G418r), YLKL593 (yku70Δ::HIS3), YLKL612 (mre11Δ::HygBr sir4Δ::LEU2), YLKL613 (mre11Δ::HygBr dnl4Δ::G418r), YLKL614 (rad50Δ::G418r
Enhancement of resistance by TLC1 occurs uniquely in mrx mutants and requires components of the homologous recombination pathway
A previous search for overexpressed yeast genes that could increase resistance of rad50 mutants to MMS, a DNA methylating agent [30] led to the isolation of library plasmids containing EXO1 and TLC1 [23]. The earlier work, in conjunction with other studies [9], revealed that the 5′-to-3′ exonuclease encoded by EXO1 can partially substitute for Mrx in processing of DSBs and increases MMS resistance by specifically elevating repair by recombination, but not NHEJ. The second suppressor gene, TLC1,
Discussion
In the current study we have identified an additional major mechanism responsible for reduction of recombinational repair of DSBs in mrx mutants. This inhibitory process was identified through an investigation of the means by which overexpression of telomerase RNA enhances resistance to DNA damage. Elevated intracellular levels of TLC1 RNA were found to increase the resistance of mrx mutants to multiple DNA damaging agents, including the S phase-dependent clastogens MMS and HU, as well as the
Conflict of interest
I have nothing to declare.
Acknowledgements
The authors wish to thank Dan Gottschling and Nancy Kleckner for gifts of plasmids and Shanna Calero for expert technical assistance. KL was supported in part by Research Corporation grant CC5767 and National Institutes of Health Grant 1R15AG028520-01A1.
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2021, Current Opinion in Genetics and DevelopmentCitation Excerpt :In organisms that predominantly use homologous recombination for DSB repair such as budding and fission yeasts, DNA-PKcs is not present and Ku is expressed at much lower levels compared to mammalian cells [23]. Nevertheless, Ku is still one of the first complexes appearing at a break site in budding yeast [24] and is still an effective block to enzymes other than MRN(X), based on work showing that deletion of Ku subunits allows for DSB 5′ processing in the absence of the MRN complex or CtIP(Ctp1/Sae2) [25•,26–29]. A competitive model for DSB repair pathway choice has derived over the years from observations that DNA ends generated in mammalian cells could be repaired by either pathway and that alteration of repair factor levels can skew repair outcomes toward NHEJ or HR [7,30].
Nej1 Interacts with Mre11 to Regulate Tethering and Dna2 Binding at DNA Double-Strand Breaks
2019, Cell ReportsCitation Excerpt :The structural features of MRX are important for end-tethering, and the endonuclease activity of Mre11 is important for HR by initiating resection at the break (Cannavo and Cejka, 2014). Ku is required for the efficient recruitment of MRX (Zhang et al., 2007); however, Ku and MRX function antagonistically in repair pathway choice (Clerici et al., 2008; Wu et al., 2008; Wasko et al., 2009; Balestrini et al., 2013). Both the CXXC hook motif and the extended coiled-coil region of Rad50 are important for DSB repair.
20 Years of Mre11 Biology: No End in Sight
2018, Molecular CellThe non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2–Sgs1 nuclease-helicase at DNA double strand breaks
2017, Journal of Biological ChemistryCitation Excerpt :Ku and MRX are believed to function antagonistically in repair pathway choice (2, 33). MRX promotes Ku removal from unrepaired DSBs (2, 38, 39), and although Ku stabilizes MRX at the break (6), Ku inhibits 5′ end degradation by MRX. Interestingly, interactions between Lif1 and Xrs2 actually enhance the binding of core NHEJ factors to Ku-bound DNA (4, 5, 40).
Interplay between Ku and replication protein A in the restriction of Exo1-mediated DNA break end resection
2015, Journal of Biological ChemistryCitation Excerpt :Taken together, these results show that Ku is quite versatile in being able to recognize a variety of DNA end structures. Yeast Ku is known to protect DSB ends from Exo1-mediated resection in cells (21, 22, 45–47), and human Ku can prevent human EXO1 from accessing blunt DNA ends in vitro (24). Human EXO1 is active on a variety of DNA structures in vitro, including nicked, gapped, and blunt-ended dsDNAs (48, 49).
Regulation of the DNA damage response by cyclin-dependent kinases
2013, Journal of Molecular BiologyCitation Excerpt :How does Cdk1-dependent phosphorylation of Sae2 promote resection at chromosome ends? In both budding and fission yeast, the DNA damage sensitivity of sae2∆ and mre11∆ cells is suppressed by KU deletion and this suppression requires both Exo1 and Sgs1,47–50 suggesting that Cdk1-dependent phosphorylation of Sae2 is needed to remove Ku from DSB ends in order to allow the action of Exo1 and Sgs1 (Fig. 1). Interestingly, removal of the Ku-mediated inhibition of resection appears to require the physical presence of MRX, but not its nuclease activity,51,52 suggesting that Ku removal does not depend on the initial processing of the DSB ends by MRX-Sae2.