DNA Repair Protein Rad55 Is a Terminal Substrate of the DNA Damage Checkpoints

Abstract

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.The SOS response in Escherichia coli provides the coordination between DNA damage sensing and the cellular responses to DNA damage (reviewed in reference 22). The primary SOS signal, single-stranded DNA (ssDNA), activates RecA in a ternary complex with ATP as a transcriptional regulator (44) and as a DNA repair protein (reviewed in reference 41). The transcriptional induction of the SOS regulon leads to increased expression of certain DNA repair genes (including RecA itself) and also elicits transient cell cycle arrest by the expression of sfiA, a cell division inhibitor (22). The activation of RecA as a repair protein leads to immediate repair of the primary damage that initiated the SOS signal. Although different in mechanism, the DNA damage checkpoints could provide a similar coordination between DNA damage sensing and repair in eukaryotes. First conceptualized as an active cell cycle control system in response to DNA damage in Saccharomyces cerevisiae (29,89), DNA damage checkpoints were later shown to control also DNA damage-induced gene expression in this organism (3). DNA damage checkpoints and DNA repair serve a common purpose to secure survival and genomic stability after DNA damage. Indirect effects of the DNA damage checkpoints on DNA repair have been discussed before (reviewed in references 18, 85, and 87), but a direct coupling of the DNA damage sensing capabilities of the checkpoint system with DNA damage repair pathways has not been identified yet.The DNA damage checkpoints in eukaryotes relay a signal in response to DNA damage to transiently delay the entry into the S or M phases, to slow down the ongoing DNA replication, or to arrest in meiotic prophase (reviewed in references 29, 62, and 87). They also elicit DNA damage-induced transcription of many genes, including some coding for DNA repair proteins (87, 93). Moreover, a related DNA replication block checkpoint ensures the dependency of M phase on a...

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…Alternatively, checkpoint inactivation might affect the stability of the Rad51 nucleoprotein filament in S/G2. Rad55 which is implicated in stabilization of Rad51 on DNA is phosphorylated in a Mec1‐Rad53‐dependent manner (Bashkirov et al , 2000) and the presence of functional Rad55/57 was reported to dictate the requirement for Srs2 in cells with DNA damage (Liu et al , 2011). Therefore, in checkpoint‐deficient cells, the lack of Rad55 phosphorylation might decrease the stability of Rad51 on DNA and promote stochastic replacement of Rad51 with RPA, thereby alleviating the need for Srs2.…”

Recombination and the Tel1 and Mec1 checkpoints differentially effect genome rearrangements driven by telomere dysfunction in yeast

“…This translocation structure is consistent with a mechanism in which broken chromosomes replicate and then fuse in inverted orientation at the broken ends by Rad51-or Rad59-dependent recombination. mec1 mutants also have a defect in Rad55 phosphorylation and reduced homologous recombination 21,30 either of which might alter the balance between the types of recombination events available to broken chromosomes. Blue indicates chromosome V, red indicates any other chromosome.…”

HDF1 and RAD17 Genes are Involved in DNA Double-strand Break Repair in Stationary Phase Saccharomyces cerevisiae

“…In haploids, the main pathway involved in the DNA damage response is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17 complex, most probably in coordination with Mec1-Rad53-Rad 55-Rad57 inducible HR [9,19]. The contribution to HR by an inducible pathway including MEC1, RAD53 and RAD 55 was indicated by other authors on the basis of molecular data.…”

“…Both sensors have orthologous protein-complexes in other eukaryotes, including man [3,[17][18][19]. MRX sensor functions both in NHEJ and homologous recombination [20][21][22].…”

“…In the haploid state the most probable DSBs repair pathway is NHEJ (MRXHdf1-Hdf2-LigIV-Lif4). In the diploid state, homologous recombination pathways depend on RAD17-MEC1-RAD53-RAD55-RAD51 gene products, as well as on MRX-Rad52 securing LH survival, and DSB repair [7,10,19]. Mec1/hATR and Tel1/hATM kinases are at the core of the network and interact with sensors at DNA damage and with downstream effectors, enabling the signal amplification [3,17,18,21,22].…”