Elsevier

DNA Repair

Volume 8, Issue 2, 1 February 2009, Pages 162-169
DNA Repair

Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae

https://doi.org/10.1016/j.dnarep.2008.09.010Get rights and content

Abstract

Yeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5′-end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was co-inactivated. Defective recombinational repair of DSBs in mrx cells thus arises from at least two separate processes: loss of Mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.

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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