Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.     

Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks

Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.     

DNA双链断裂的非同源末端连接修复

细胞内普遍存在的DNA双链断裂(DSB)可通过同源重组(HR)或非同源末端连接(NHEJ)修复。由于HR仅在存在相同染色体作为模板的时候进行,因此,NHEJ通常为主要的修复方式。在NHEJ中,DSB末端首先由Ku识别,接着由核酸酶、聚合酶在Ku与DNA-PKcs协助下加工,并由连接酶IVXRCC4-XLF连接。NHEJ底物类型多样,末端的修复常包含反复加工的过程,导致修复产物通常无法复原损伤前的序列。虽然无法确保准确修复DNA,NHEJ仍对维持基因组的稳定性具有重要的意义。对NHEJ的研究有助于理解癌症的发生机制并将促进癌症的治疗。     

The mechanism of human nonhomologous DNA end joining

Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G(2), whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time.     

Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.     

Xlf1 is required for DNA repair by nonhomologous end joining in Schizosaccharomyces pombe

The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins.     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

Role of RecA in DNA damage repair in Deinococcus radiodurans

It has been shown previously that the RecA protein of Deinococcus radiodurans plays a unique role in the repair of DNA damage in this highly DNA damage-resistant organism. Despite the high level of amino-acid identity, previous work has shown that Escherichia coli RecA does not complement D. radiodurans RecA mutants, further suggesting the uniqueness of D. radiodurans RecA. The work presented here shows that E. coli RecA does in fact provide partial complementation to a D. radiodurans RecA null mutant, suggesting that the RecA protein from D. radiodurans may not be as unique as believed previously.     

Mechanisms of overlap formation in nonhomologous DNA end joining.

Rejoining of nonhomologous DNA termini plays a central role in processes of illegitimate recombination. In Xenopus egg extracts, DNA ends with noncomplementary 4-nucleotide antiparallel single-strand protrusions are assumed to be joined by formation of short mismatched overlap intermediates. The extents of these overlaps may be set by single fortuitously matching base pairs and determine the patterns of subsequent gap filling and nick ligation. Under conditions of alternative overlap settings, rules for the most probable joining pathway and the effects of mismatches on junction formation were analyzed. We show that in certain cases, fill-in and ligation converting overlap intermediates into covalently closed junctions may proceed in the presence of unrepaired mismatches, whereas in other cases, completion of junction formation is preceded by removal of mismatches. Results are discussed in relation with "alignment" proteins postulated to structurally support overlap heteroduplexes during junction formation.     

耐辐射球菌DNA双链断裂修复机制研究新进展

耐辐射球菌对于电离辐射等DNA损伤剂具有极强的抗性,能够将同一个基因组中同时产生的高达100个以上的DNA双链断裂在数十小时内高效而精准地进行修复,是研究DNA双链断裂修复机制的重要模式生物。同源重组、非同源末端连接和单链退火途径作为3个主要的修复途径参与了耐辐射球菌基因组DNA双链断裂的修复过程。此外,一系列新发现的重要蛋白质,如Ppr I、Ddr B等对于耐辐射球菌基因组的修复过程同样至关重要。根据本实验室和国内外在这一研究领域近年来的报道,以不同的修复途径为线索,综述该菌DNA双链断裂修复机制的最新研究成果。     

RecO is essential for DNA damage repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radiodurans's radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.     

The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining

Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.     

Involvement of the nonhomologous end joining DNA repair pathway in the bystander effect for chromosomal aberrations

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.     

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.     

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.     

DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1 < S < G2/M). HR is nearly absentin G1, most active in the S phase, and declines in G2/M. Thus, inG2/M NHEJ is elevated, while HR is on decline. This is in contrastto a general belief that NHEJ is most active in G1, while HR isactive in S, G2 and M. The overall efficiency of NHEJ was higherthan HR at all cell cycle stages. We conclude that human somaticcells utilize error-prone NHEJ as the major DSB repair pathway atall cell cycle stages, while HR is used, primarily, in the S phase.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

Deinococcus radiodurans YgjD and YeaZ are involved in the repair of DNA cross-links

Orthologs of Escherichia coli ygjD and yeaZ genes are highly conserved in various organisms. The genome of the radioresistant bacterium Deinococcus radiodurans possesses single orthologs of ygjD (DR_0382) and yeaZ (DR_0756). Complete loss of either one or both genes did not result in any significant changes in cell growth efficiency, indicating that both genes are not essential for cell viability in D. radiodurans, unlike the case with other species such as E. coli, Bacillus subtilis and Saccharomyces cerevisiae. Survival rates following DNA damage induced by hydrogen peroxide (H₂O₂), N-methyl-N??-nitro-N-nitrosoguanidine (MNNG), ultra violet (UV) radiation, ??-rays, cisplatin and mitomycin C (MMC) were compared among the wild-type strain and D. radiodurans ygjD/yeaZ null mutants. Cell viability of the null mutants did not decrease following exposure to H₂O₂ or MNNG. In addition, the reduction in cell viability following exposure to ??-rays, UV radiation or cisplatin was marginal in the null mutants compared to the wild-type strain. Interestingly, the null mutants exhibited high sensitivity to MMC, which mainly causes interstrand DNA cross-links. The sensitivity of the null mutants to MMC was restored to that of the wild type by transformation with plasmids expressing these genes. These results suggest that D. radiodurans ygjD and yeaZ genes are involved in DNA repair and play a role in the repair of DNA cross-links.