Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.     

Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes

Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents. An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation. To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes. This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes. Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide. To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable. Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination. Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA. We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.     

Effects of HDF1 (Ku70) and HDF2 (Ku80) on spontaneous and DNA damage-induced intrachromosomal recombination in Saccharomyces cerevisiae

The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining. HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination. Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear. We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination). We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination. As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing. The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination. This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.     

Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.

S. cerevisiae RAD50, MRE11, and XRS2 genes are required for telomere maintenance, cell cycle checkpoint signaling, meiotic recombination, and the efficient repair of DNA double-strand breaks (DSB)s by homologous recombination and nonhomologous end-joining (NHEJ). Here, we demonstrate that the complex formed by Rad50, Mre11, and Xrs2 proteins promotes intermolecular DNA joining by DNA ligase IV (Dnl4) and its associated protein Lif1. Our results show that the Rad50/Mre11/Xrs2 complex juxtaposes linear DNA molecules via their ends to form oligomers and interacts directly with Dnl4/Lif1. We also demonstrate that Rad50/Mre11/Xrs2-mediated intermolecular DNA joining is further stimulated by Hdf1/Hdf2, the yeast homolog of the mammalian Ku70/Ku80 heterodimer. These studies reveal specific functional interplay among the Hdf1/Hdf2, Rad50/Mre11/Xrs2, and Dnl4/Lif1 complexes in NHEJ.     

Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways.

Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.     

Regulation of Rad51 function by phosphorylation

Rad51 is a key enzyme involved in DNA double-strand break repair by homologous recombination. Here, we show that in response to DNA damage, budding yeast Rad51 is phosphorylated on Ser 192 in a manner that is primarily mediated by the DNA-damage-responsive protein kinase Mec1. We show that mutating Rad51 Ser 192 to Ala or Glu confers hypersensitivity to DNA damage and homologous-recombination defects. Furthermore, biochemical analyses indicate that Ser 192 is required for Rad51 adenosine triphosphate hydrolysis and DNA-binding activity in vitro, whereas mutation of Ser 192 does not interfere with Rad51 multimer formation. These data suggest a model in which Mec1-mediated phosphorylation of Rad51 Ser 192 in response to DNA damage controls Rad51 activity and DNA repair by homologous recombination.     

Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid

We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis. Received: 30 October 1996 / Accepted: 28 February 1997     

Interaction with Rad51 is indispensable for recombination mediator function of Rad52

In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.     

Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes

The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.     

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.     

Role of DnaB helicase in UV-induced illegitimate recombination in Escherichia coli

To study the involvement of DNA replication in UV-induced illegitimate recombination, we examined the effect of temperature-sensitive dnaB mutations on illegitimate recombination and found that the frequency of illegitimate recombination was reduced by an elongation-deficient mutation, dnaB14, but not by an initiation-deficient mutation, dnaB252. This result indicates that DNA replication is required for UV-induced illegitimate recombination. In addition, the dnaB14 mutation also affected spontaneous or UV-induced illegitimate recombination enhanced by the recQ mutation. Nucleotide sequence analyses of the recombination junctions showed that DnaB-mediated illegitimate recombination is short homology dependent. Previously, Michel et al. (B. Michel, S. Ehrlich, and M. Uzest, EMBO J. 16:430--438, 1997) showed that thermal treatment of the temperature-sensitive dnaB8 mutant induces double-stranded breaks, implying that induction of illegitimate recombination occurs. To explain the discrepancy between the observations, we propose a model for DnaB function, in which the dnaB mutations may exhibit two types of responses, early and late responses, for double-stranded break formation. In the early response, replication forks stall at damaged DNA, resulting in the formation of double-stranded breaks, and the dnaB14 mutation reduces the double-stranded breaks shortly after temperature shift-up. On the other hand, in the late response, the arrested replication forks mediated by the dnaB8 mutation may induce double-stranded breaks after prolonged incubation.     

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.     

Type I restriction enzyme with RecA protein promotes illegitimate recombination

Illegitimate (non-homologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. However, we have found a type of illegitimate recombination that requires an interaction between long homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type I (EcoKI) restriction in vivo within a special mutant strain of Escherichia coli. In the present work, we analyzed genetic requirements for this type of illegitimate recombination in well-defined genetic backgrounds. Our analysis demonstrated dependence on RecA function and on the presence of two EcoKI sites on the substrate DNA. These results are in harmony with a model in which EcoKI restriction enzyme attacks an intermediate of homologous recombination to divert it to illegitimate recombination.     

Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair.

To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.     

A physiological significance of the functional interaction between Mus81 and Rad27 in homologous recombination repair

Fen1 and Mus81–Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81_Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81–Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21–26 (aa from 21–26) and N108–114 (aa from 108–114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures.