The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death. Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination. We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle. We show that cells arrested at the G1 phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ. Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.     

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

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Interplay of two major repair pathways in the processing of complex double-strand DNA breaks

Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.     

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (gammaH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, gammaH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.     

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair.     

The changes of DNA double-strand breaks and DNA repair during ovarian reserve formation in mice

DNA double-strand break (DSB) repair is crucial to maintain genomic stability for sufficient ovarian reserve. It remains unknown the changes of DSBs formation and DNA repair in germ cells during ovarian reserve formation in FVB/N mice. We demonstrated germ cell numbers increased significantly (all P < 0.05) from E11.5 to E13.5 and decreased significantly (all P> 0.05) until P2. OCT4 and SOX2 analyses indicated pluripotency peaks at E13.5 then decreases significantly (all P 0.05) until P2. γH2AX analyses revealed DSB formation significantly (P < 0.05) increased from E13.5 until P2. RAD51 and DMC1 data revealed homologous recombination (HR) pathway repair of DSBs is persistent active during meiosis (E13.5- P2) (all P> 0.05). 53BP1 and KU70 data indicate the non-homologous end-joining pathway (NHEJ) remains active during meiosis. 53BP1 expression was highest at E13.5 (P < 0.05). KU70 expression was higher in germ cells from E15.5 to P2 (all < P 0.05). PH3 and KI67 analyses revealed germ cell proliferation was not significantly different (all P> 0.05) from E13.5 to P2. Caspase-3 and TUNEL analyses showed germ cells apoptosis was not significantly different (all P > 0.05) from E13.5 to P2. In conclusion, we found both germ cell number and pluripotency peak at E13.5 and decline during meiosis. We demonstrated HR and NHEJ continually repair DSBs during meiosis. RAD51 and DMC1 are continuously expressed during meiosis. 53BP1 is mainly expressed at E13.5. KU70 continually functions from E15.5 to P2. Proliferating and apoptotic cells were rarely detected during meiosis. Results provide a basis for further study of how DSBs and DNA repair affect germ cell development.     

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

Choreography of recombination proteins during the DNA damage response

Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.     

The homologous chromosome is an effective template for the repair of mitotic DNA double-strand breaks in Drosophila

In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining.     

A nanomachine for making ends meet: MRN is a flexing scaffold for the repair of DNA double-strand breaks

In a recent study (Moreno-Herrero et al., 2005), atomic force microscopy (AFM) imaging of the human Mre11/Rad50/Nbs1 (MRN) complex engaging substrate DNA revealed large-scale, DNA binding-induced propagation of conformational change to the distal ends of the Rad50 coiled coils and erection of a 1000 A scaffold to productively bridge DNA ends.     

Effect of Wortmannin on the repair profiles of DNA double-strand breaks in the whole genome and in interstitial telomeric sequences of Chinese hamster cells

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process.     

Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes

Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers (COs) are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover (NCO) recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, it is becoming increasingly clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically-proliferating cells and that the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires COs) than mitotic recombination (repair of DNA damage, which typically generates NCOs).     

The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results.     

Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome

Different DNA repair pathways that use homologous sequences in close proximity to genomic double-strand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the efficiency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the beta-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about five times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most efficient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a significant role in the evolution of tandemly arranged gene families like resistance genes.     

The Drosophila hus1 gene is required for homologous recombination repair during meiosis

The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Previously, we demonstrated that Drosophila hus1 is essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. In this study, we further analyzed the function of hus1 during meiosis and discovered that the synaptonemal complex (SC) disassembles abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, we showed that in hus1, DSBs that form during meiosis are not processed efficiently, and that this defect is not suppressed by a mutation in DmChk2. We found a genetic interaction between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination.     

Delineation of a minimal topoisomerase II binding protein 1 for regulated activation of ATR at DNA double-strand breaks

Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein–protein and protein–DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1’s nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 “Junior” and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.     

A tale of tails: insights into the coordination of 3′ end processing during homologous recombination

Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1–Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3′ single‐stranded intermediates formed during single‐strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1–Rad10‐dependent removal of 3′ nonhomologous tails during homologous recombination, including Msh2–Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1–Rad10‐dependent end processing is coordinated: Msh2–Msh3 stabilizes and prepares double‐strand/single‐strand junctions for Rad1–Rad10 cleavage, Saw1 recruits Rad1–Rad10 to 3′ tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1–Rad10.