原核生物的NHEJ修复途径

DNA双链断裂(DSBs)是严重的DNA损伤形式之一,生物体对DSBs的修复可通过同源重组(HR)或非同源末端连接途径(NHEJ)进行。长期以来,人们普遍认为HR是细菌DSBs修复的惟一途径,但在分支杆菌和其它原核生物体内NHEJ途径的发现,使这一观念得以颠覆。最近的研究表明,细菌NHEJ修复系统是一个双组分系统,包含一个多功能的DNA连接酶(LigD)和DNA末端结合蛋白Ku,具有DSBs修复所需的断裂末段识别、末端加工和连接活性。重点综述细菌NHEJ修复系统的组成、结构以及生理功能。     

The function of classical and alternative non‐homologous end‐joining pathways in the fusion of dysfunctional telomeres

Repair of DNA double‐stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non‐homologous end‐joining (NHEJ) or error‐free homologous recombination. NHEJ precedes either by a classic, Lig4‐dependent process (C‐NHEJ) or an alternative, Lig4‐independent one (A‐NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere‐binding proteins are recognized as DSBs. In this report, we studied which end‐joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end‐to‐end chromosome fusions mediated by the C‐NHEJ pathway. In contrast, removal of Tpp1–Pot1a/b initiated robust chromosome fusions that are mediated by A‐NHEJ. C‐NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A‐NHEJ is a major pathway to process dysfunctional telomeres.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.     

Bacterial DNA repair by non-homologous end joining

The capacity to rectify DNA double-strand breaks (DSBs) is crucial for the survival of all species. DSBs can be repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). The long-standing notion that bacteria rely solely on HR for DSB repair has been overturned by evidence that mycobacteria and other genera have an NHEJ system that depends on a dedicated DNA ligase, LigD, and the DNA-end-binding protein Ku. Recent studies have illuminated the role of NHEJ in protecting the bacterial chromosome against DSBs and other clastogenic stresses. There is also emerging evidence of functional crosstalk between bacterial NHEJ proteins and components of other DNA-repair pathways. Although still a young field, bacterial NHEJ promises to teach us a great deal about the nexus of DNA repair and bacterial pathogenesis.     

Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells

DNA double-strand breaks (DSBs) may be caused by normal metabolic processes or exogenous DNA damaging agents and can promote chromosomal rearrangements, including translocations, deletions, or chromosome loss. In mammalian cells, both homologous recombination and nonhomologous end joining (NHEJ) are important DSB repair pathways for the maintenance of genomic stability. Using a mouse embryonic stem cell system, we previously demonstrated that a DSB in one chromosome can be repaired by recombination with a homologous sequence on a heterologous chromosome, without any evidence of genome rearrangements (C. Richardson, M. E. Moynahan, and M. Jasin, Genes Dev., 12:3831-3842, 1998). To determine if genomic integrity would be compromised if homology were constrained, we have now examined interchromosomal recombination between truncated but overlapping gene sequences. Despite these constraints, recombinants were readily recovered when a DSB was introduced into one of the sequences. The overwhelming majority of recombinants showed no evidence of chromosomal rearrangements. Instead, events were initiated by homologous invasion of one chromosome end and completed by NHEJ to the other chromosome end, which remained highly preserved throughout the process. Thus, genomic integrity was maintained by a coupling of homologous and nonhomologous repair pathways. Interestingly, the recombination frequency, although not the structure of the recombinant repair products, was sensitive to the relative orientation of the gene sequences on the interacting chromosomes.     

Developmental modulation of nonhomologous end joining in Caenorhabditis elegans

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.     

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.     

RIF1: A novel regulatory factor for DNA replication and DNA damage response signaling

DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5′–3′ end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity.     

Development of a rapid,small-scale DNA repair assay for use on clinical samples

Double-strand breaks (DSBs) are the most lethal form of DNA damage. They can be repaired by one of two pathways, homologous recombination and non-homologous end joining (NHEJ). A NHEJ assay has previously been reported which measures joining using cell-free extracts and a linearised plasmid as DNA substrate. This assay was designed for 3 × 10⁹ cells grown in vitro and utilised radioactively labelled substrate. We have scaled down the method to use smaller cell numbers in a variety of cell lines. Altering the cellular extraction procedure decreased background DNA contamination. The cleaner preparations allowed us to use SYBR Green I staining to identify joined products, which was as sensitive as ³²P-end-labelled DNA. NHEJ was found in established tumour cell lines from different originating tissues, though actual levels and fidelity of repair differed. This method also allowed end joining to be assessed in clinical specimens (human blood, brain and bladder tumours) within 24 h of receiving samples. The application of this method will allow investigation of the role of DSB DNA repair pathways in human tumours.     

CRISPR with a Happy Ending: Non‐Templated DNA Repair for Prokaryotic Genome Engineering

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR‐Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR‐Cas and other nucleases mediate double‐strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template‐independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non‐homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.     

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2-/- and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.     

MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

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。     

WRN,the protein deficient in Werner syndrome,plays a critical structural role in optimizing DNA repair

Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3' --> 5' exonuclease and 3' --> 5' helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN-/-) complemented with either wtWRN, exonuclease-defective WRN (E-), helicase-defective WRN (H-) or exonuclease/helicase-defective WRN (E-H-). The single E-and H- mutants each partially complemented the NHEJ abnormality of WRN-/- cells. Strikingly, the E-H- double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E- and H- single mutants increased HR to levels higher than those restored by either E-H- or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events.     

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was "blunt-end ligation" subsequent to "fill-in" or "blunting", with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.     

Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1

DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway.     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

KAP1 Deacetylation by SIRT1 Promotes Non-Homologous End-Joining Repair

Homologous recombination and non-homologous end joining are two major DNA double-strand-break repair pathways. While HR-mediated repair requires a homologous sequence as the guiding template to restore the damage site precisely, NHEJ-mediated repair ligates the DNA lesion directly and increases the risk of losing nucleotides. Therefore, how a cell regulates the balance between HR and NHEJ has become an important issue for maintaining genomic integrity over time. Here we report that SIRT1-dependent KAP1 deacetylation positively regulates NHEJ. We show that up-regulation of KAP1 attenuates HR efficiency while promoting NHEJ repair. Moreover, SIRT1-mediated KAP1 deacetylation further enhances the effect of NHEJ by stabilizing its interaction with 53BP1, which leads to increased 53BP1 focus formation in response to DNA damage. Taken together, our study suggests a SIRT1-KAP1 regulatory mechanism for HR-NHEJ repair pathway choice.