Yeast DNA ligase IV mediates non-homologous DNA end joining

The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination. Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions. Here we show that S. cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 and the previously unknown DNA ligase IV homologue DNL4. dnl4 mutants are deficient in precise and end-processed NHEJ. DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects. dnl4 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation. Thus, S. cerevisiae has two distinct and separate ligation pathways.     

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].     

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.     

Maximum pulsed electromagnetic field limits based on peripheral nerve stimulation: application to IEEE/ANSI C95.1 electromagnetic field standards

DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.     

Cell-free V(D)J recombination

V(D)J recombination generates diversity in the immune system through the lymphoid-specific assembly of multiple gene segments into functional immunoglobulin and T-cell receptor genes. The first step in V(D)J recombination is cleavage of DNA at recombination signal sequences. Cleavage produces a blunt DNA end on each signal sequence and a hairpin end on adjacent coding gene segments, and can be reproduced in vitro by using purified RAG and RAG2 proteins. The later steps involve processing and joining of the cleaved DNA ends, and until now have been studied only in cells. Here we reconstitute the complete V(D)J recombination reaction in a cell-free system. We find that the RAG proteins are not only involved in cleavage, but are also needed in the later steps for efficient joining of coding ends. Joining is largely directed by short pieces of identical sequence in the coding flanks, but addition of human DNA ligase I results in greater diversity. Coding junctions contain short deletions as well as additions complementary to a coding flank (P nucleotides). Addition of non-templated nucleotides into coding junctions is mediated by terminal deoxyribonucleotidyl transferase. The cell-free reaction can therefore reproduce the complete set of processing events that occur in cells.     

A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events

Eukaryotic repair of double-strand DNA breaks can occur either by homologous recombination or by nonhomologous DNA end joining (NHEJ). NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and DNA-dependent protein kinase. NHEJ involves a synapsis step in which the two ends are maintained in proximity, processing steps in which nucleases and polymerases act on the ends, an alignment step in which a few nucleotides of terminal homology guide the ends into preferred alignments, and a ligation step. Some of the steps, such as ligation, rely on a single enzymatic component. However, the processing steps begin and end with a wide array of alternative substrates and products, respectively, and likely involve multiple nucleases and polymerases. Given the alternative pathways that can be catalyzed by the remaining nucleases and polymerases, no one of these processing enzymes is likely to be essential. The only requirement for the processing enzymes, as a collective, is to generate a ligatable configuration, namely a ligatable nick on each strand. Here, we have tested the two major known 5'-specific nucleases of Saccharomyces cerevisiae for involvement in NHEJ. Whereas EXO1 does not appear to be involved to any detectable level, deleting RAD27 (FEN-1 of yeast) leads to a 4.4-fold reduction specifically of those NHEJ events predicted to proceed by means of 5' flap intermediates. Because Rad27/FEN-1 acts specifically at 5' flap structures, these results suggest that the NHEJ alignment step precedes nucleolytic processing steps in a significant fraction of NHEJ events.     

DNA end-joining catalyzed by human cell-free extracts

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.     

Supplementation of MCF-7 cells with essential fatty acids induces the activation of protein kinase C in response to IGF-1

V(D)J recombination consists of a DNA cleavage reaction catalysed by RAG1 and RAG2, followed by an end-joining reaction that utilizes the cell's double-strand break repair machinery. Genes essential for the end-joining reaction include: XRCC4 encoding a protein of unknown enzymatic function; XRCC5 and XRCC6 encoding 86 and 70 kDa subunits of the Ku autoantigen, a DNA end-binding protein that is also the regulatory subunit of DNA-dependent protein kinase (DNA-PK); and XRCC7 encoding the catalytic subunit (DNA-PKcs) of DNA-PK. Recent progress in understanding the cleavage reaction, coupled with what was previously known about Ku, DNA-PK, and double-strand break repair, provide the foundation for a working model of how V(D)J recombination might be catalysed.     

Cryptic signals and the fidelity of V(D)J joining

V(D)J recombination is responsible for the de novo creation of antigen receptor genes in T- and B-cell precursors. To the extent that lymphopoiesis takes place throughout an animal's lifetime, recombination errors present an ongoing problem. One type of aberrant rearrangement ensues when DNA sequences resembling a V(D)J joining signal are targeted by mistake. This study investigates the type of sequence likely to be subject to mistargeting, the level of joining-signal function associated with these sequences, and the number of such cryptic joining signals in the genome.     

Double-strand break repair in Ku86- and XRCC4-deficient cells

The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.     

P nucleotides, hairpin DNA and V(D)J joining: making the connection

The fine-structures of V(D)J joining products present a cache of information about the recombination process. Testable hypotheses with predictive value are beginning to provide a surprisingly detailed picture of the joining operation. Here, one feature of V(D)J junctions, the appearance of short palindromic inserts, is discussed, and experimental evidence in support of a joining model involving a hairpin-terminated DNA intermediate will be summarized.     

Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA

Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.     

Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.     

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-alpha forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-beta, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

The role of maternal hostility and family environment upon cardiovascular functioning among youth two years later: socioeconomic and ethnic differences

The DNA-dependent protein kinase (DNA-PK) consists of Ku70, Ku80, and a large catalytic subunit, DNA-PKcs. Targeted inactivation of the Ku70 or Ku80 genes results in elevated ionizing radiation (IR) sensitivity and inability to perform both V(D)J coding-end and signal (RS)-end joining in cells, with severe growth retardation plus immunodeficiency in mice. In contrast, we now demonstrate that DNA-PKcs-null mice generated by gene-targeted mutation, while also severely immunodeficient, exhibit no growth retardation. Furthermore, DNA-PKcs-null cells are blocked for V(D)J coding-end joining, but retain normal RS-end joining. Finally, while DNA-PK-null fibroblasts exhibited increased IR sensitivity, DNA-PKcs-deficient ES cells did not. We conclude that Ku70 and Ku80 may have functions in V(D)J recombination and DNA repair that are independent of DNA-PKcs.     

Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.     

Failure of hairpin-ended and nicked DNA To activate DNA-dependent protein kinase: implications for V(D)J recombination

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.     

Nonhomologous recombination in human cells

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.     

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.