Deletion of yeast TPK1 reduces the efficiency of non-homologous end joining DNA repair

https://doi.org/10.1016/j.bbrc.2020.09.083Get rights and content

Highlights

  • Protein-protein interaction analysis suggests a new role for human PRKACB in NHEJ.

  • Deletion of TPK1, the yeast homolog of PRKACB reduces the efficiency of the plasmid and chromosomal break repairs in yeast.

  • Genetic interaction analysis further connects a role for TPK1 to DNA break repair.

  • The activity of TPK1 seems to be independent of the YKU complex.

Abstract

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.

Introduction

DNA double stranded breaks (DSBs) are the most severe forms of DNA damage. DSBs are induced either endogenously by replication errors and metabolic oxidative products or as a result of exogenous reagents such as environmental stress, radiation or toxic chemicals [1]. Once the phosphodiester bond of both strands is damaged, the DNA will be divided into two fragments. This fragmentation can cause genomic instability and chromosomal rearrangement [2]. Mutations arising from mis-repaired or unrepaired DSBs can lead to the onset of complex diseases such as cancer, neurodegeneration and immunodeficiency [2]. DSBs are primarily repaired by two pathways that work independently: homologous recombination (HR) and non-homologous end joining (NHEJ). HR uses homologous templates usually obtained from sister chromatids or homologous chromosomes to connect the broken sites of DSBs, producing an error-free repair [3]. This pathway is preferred in cases of stalled replication forks [3]. DSBs can also be ligated directly through NHEJ pathway in an error-prone manner [4]. NHEJ is the main repair pathway in human cells and is highly conserved from simple eukaryotes such as the yeast, Saccharomyces cerevisiae, to a more complex mammalian system [1,2,4].

The process of NHEJ involves three main protein complexes for repair initiation, ligation preparation and, finally, for the rejoining of the broken ends. The first protein complex to initiate NHEJ is Yku70/Yku80, which attaches to the broken ends of DNA and recruits other repair factors to the site of damage [5]. Recognition of the damage and recruitment of the YKU complex to the site of damage involves a cascade of DNA damage checkpoints in which Tel1, Mec1 and Rad53 play central roles [6]. Once YKU is bound to the DNA ends, it recruits the MRX complex (Mre11, Rad50 and Xrs2) to the site of damage [5]. In preparation for ligation, the MRX complex forms a bridge between the broken ends and brings them in close proximity [7]. The final ligation of the DSB requires Lif1/Lig4 complex. Lif1 is brought to the break site by YKU and subsequently recruits Lig4, a protein with ligase activity [8]. Another factor suggested as part of Lif1/Lig4 complex is Nej1 due to its interaction with Lif1 [9]. Even though the interactions within the core components of NHEJ are well understood, several other proteins have been shown to play a role in NHEJ such as Rtt109 [10], Pol3 [11], Tdp1 [12] and Pph3/Psy2 [13], suggesting existence of other proteins in the NHEJ pathway.

Kinases often play a central role in signal transduction and protein networking within a pathway and between different pathways in the cell. Protein kinase A (PKA) is one of the primary kinases functioning in many cellular processes such as metabolism, stress response, nutrient starvation and the cell cycle [14]. In yeast cells, PKA has three catalytic protein subunits, Tpk1, Tpk2 and Tpk3 [14]. Here we report a role for Tpk1 in DSB repair through NHEJ. Our data shows that the deletion of TPK1 shows sensitivity to DNA damaging agents. Additionally, TPK1 mutant strains significantly reduce the efficiency of NHEJ repair in both plasmid and chromosomal break repair assays.

Section snippets

Yeast strains and plasmids

Yeast mutant strains were obtained from S. cerevisiae deletion library in BY4741 background (MATa orfΔ:KanMX4 his3Δ leu2Δ met15Δ ura3Δ) [15]. TPK1 was deleted in Y7092 (MATa can1Δ:STE2pr-HIS3 lyp11Δ ura31Δ leu21Δ his31Δ met151Δ) and TPK1 and YKU80 were deleted in JKM139 (MATa hmrΔ:ADE1 hmlΔ:ADE1 ade1-100 leu2-3112 lys5 trp1:hisG ura3-52 ade3:GAL-HO) background [16], by PCR transformation method as in Ref. [15] which replaces the target gene with a NatR and/or HygR cassette. Plasmids p416, which

Identification of TPK1 as a candidate involved in double stranded DNA break repair

Protein-protein interaction (PPI) analysis has been extensively used to predict new function(s) and to better understand the function(s) of various proteins [23]. PPIs can be studied various experimental methods such as affinity purification and yeast two hybrid as well as computational approaches [24]. We have previously developed a computational method to predict de novo PPIs in different organisms and most recently in humans [20,25]. In the current study we used our computational approach,

Concluding remarks

Kinases are important regulators of protein activities. They participate in regulation of the majority of cellular processes including DNA damage repair. Kinases such as Dun1, Rad53, Tel1 and Mec1 are key regulators of DNA repair pathways, playing important roles in directing the cell to a specific repair pathway and in the recruitment of repair proteins [37]. Here, we implicated a role for TPK1 in repair of blunt end and sticky end DSBs via NHEJ mechanism. It is possible that TPK1 exerts its

Declaration of competing interest

All authors declare that they don’t have any conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgement

This work is dedicated to the memory of our friend and colleague Fareed Arasteh, who lost his life in the flight 752 Plane Crash, 2020.

References (37)

  • L. Deriano et al.

    Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage

    Annu. Rev. Genet.

    (2013)
  • L.S. Symington et al.

    Double-strand break end resection and repair pathway choice

    Annu. Rev. Genet.

    (2011)
  • K. K Chiruvella et al.

    Repair of double-strand breaks by end joining

    Cold Spring Harb Perspect Biol

    (2013)
  • D. Wu et al.

    Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in saccharomyces cerevisiae

    Genetics

    (2008)
  • P.L. Palmbos et al.

    Recruitment of saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain

    Genetics

    (2008)
  • K. Bahmed et al.

    Yeast Tdp1 regulates the fidelity of nonhomologous end joining

    Proc. Natl. Acad. Sci. Unit. States Am.

    (2010)
  • K. Omidi et al.

    Phosphatase complex Pph3/Psy2 is involved in regulation of efficient non-homologous end-joining pathway in the yeast saccharomyces cerevisiae

    PloS One

    (2014)
  • E.A. Winzeler et al.

    Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis

    Science

    (1999)
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