KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion

The KARP-1 (Ku86 Autoantigen Related Protein-1) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.

Association of terminal deoxynucleotidyl transferase with Ku

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein–protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

Replication-dependent early meiotic requirement for Spo11 and Rad50

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11–1 or rad50–4 and another mutant, spo22–1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Tid1/Rdh54 promotes colocalization of Rad51 and Dmc1 during meiotic recombination

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also contain a tid1/rdh54 mutation. The role of Tid1/Rdh54 in coordinating RecA homolog assembly may be very direct, because Tid1/Rdh54 is known to physically bind both Dmc1 and Rad51. Also, Dmc1 foci appear early in a tid1/rdh54 mutant. Thus, Tid1 may normally act with Rad51 to promote ordered RecA homolog assembly by blocking Dmc1 until Rad51 is present. Finally, whereas double-staining foci predominate in WT nuclei, a subset of nuclei with expanded chromatin exhibit individual Rad51 and Dmc1 foci side-by-side, suggesting that a Rad51 homo-oligomer and a Dmc1 homo-oligomer assemble next to one another at the site of a single double-strand break (DSB) recombination intermediate.

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3Cdc34 goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.

RAD50 function is essential for telomere maintenance in Arabidopsis

We have identified and characterized an Arabidopsis thaliana rad50 mutant plant containing a T-DNA insertion in the AtRAD50 gene and showing both meiotic and DNA repair defects. We report here that rad50/rad50 mutant cells show a progressive shortening of telomeric DNA relative to heterozygous rad50/RAD50 controls and that the mutant cell population rapidly enters a crisis, with the majority of the cells dying. Surviving rad50 mutant cells have longer telomeres than wild-type cells, indicating the existence in plants of an alternative RAD50-independent mechanism for telomere maintenance. These results report the role of a protein essential for double-strand break repair in telomere maintenance in higher eukaryotes.

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.

The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability

Many DNA polymerases (Pol) have an intrinsic 3′→5′ exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3′→5′ Exo of Pol δ as a supplement or backup for the Rad27/Fen1 5′ flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol δ mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol δ. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol δ Exo I and Exo II motifs. However, rad27-p Pol δ Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5′ flaps in the lagging strand. These results suggest that the 3′→5′ Exo activity of Pol δ is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.

Direct DNA binding by Brca1

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein–DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Rescue of arrested replication forks by homologous recombination

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

RecA protein promotes the regression of stalled replication forks in vitro

Replication forks are halted by many types of DNA damage. At the site of a leading-strand DNA lesion, forks may stall and leave the lesion in a single-strand gap. Fork regression is the first step in several proposed pathways that permit repair without generating a double-strand break. Using model DNA substrates designed to mimic one of the known structures of a fork stalled at a leading-strand lesion, we show here that RecA protein of Escherichia coli will promote a fork regression reaction in vitro. The regression process exhibits an absolute requirement for ATP hydrolysis and is enhanced when dATP replaces ATP. The reaction is not affected by the inclusion of the RecO and R proteins. We present this reaction as one of several potential RecA protein roles in the repair of stalled and/or collapsed replication forks in bacteria.

Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F′ plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′ lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.