XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3' overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1-4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.     

Nonhomologous DNA end joining in Schizosaccharomyces pombe efficiently eliminates DNA double-strand-breaks from haploid sequences.

Cells of higher eucaryotes are known to possess mechanisms of illegitimate recombination which promote the joining between nonhomologous ends of broken DNA and thus may serve as basic tools of double-strand-break (DSB) repair. Here we show that cells of the fission yeast Schizosaccharomyces pombe also contain activities of nonhomologous DNA end joining resembling the ones found in higher eucaryotes. Nonhomologous end joining activities were detected by transformation of linearized self-replicating plasmids in yeast cells employing a selection procedure which only propagates transformants carrying recircularized plasmid molecules. Linear plasmid substrates were generated by duplicate restriction cuts carrying either blunt ends or 3' or 5' protruding single strands (PSS) of 4 nt which were efficiently joined in any tested combination. Sequence analysis of joined products revealed that junctional sequences were shortened by 1 to 14 nt. Two mechanisms may account for junction formation (i) loss of terminal nucleotides from PSS tails to produce blunt ends which can be joined to abutting ends and (ii) interactions of DNA termini at patches of sequence homologies (1-4 bp) by formation of overlap intermediates which are subsequently processed. A general feature of the yeast joining system is that end joining can only be detected in the absence of sequence homology between the linear substrate and host genome. In the presence of homology, nonhomologous DNA end joining is efficiently competed by activities of homologous recombination.     

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.     

A novel pathway of DNA end-to-end joining

Repair mechanisms related to illegitimate recombination can join nonhomologous DNA ends that terminate as protruding single strands (PSS). Here we analyze in Xenopus egg extracts joining reactions between 3' PSS termini and various partner termini. In junctions, 3' PSS termini are preserved by fill-in DNA synthesis, although their 5' recessed ends cannot serve as a primer. Alternative priming from a partner terminus ligated to the 3' PSS end appears unlikely, because no single strand-specific DNA ligases are detectable. We show that fill-in of 3' PSS termini precedes ligation and can even be primed in the absence of any ligation. Therefore, priming requires precise alignment of terminus pairs by a novel mechanism. We postulate that this is achieved by unique DNA binding proteins that align ends in various types of joining reactions.     

Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.     

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.     

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.     

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。     

Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK), consisting of Ku and the DNA-PK catalytic subunit (DNA-PKcs), and the DNA ligase IV-XRCC4 complex function together in the repair of DNA double-strand breaks by non-homologous end joining. These protein complexes are also required for the completion of V(D)J recombination events in immune cells. Here we demonstrate that the DNA ligase IV-XRCC4 complex binds specifically to the ends of duplex DNA molecules and can act as a bridging factor, linking together duplex DNA molecules with complementary but non-ligatable ends. Although the DNA end-binding protein Ku inhibited DNA joining by DNA ligase IV-XRCC4, it did not prevent this complex from binding to DNA. Instead, DNA ligase IV-XRCC4 and Ku bound simultaneously to the ends of duplex DNA molecules. DNA ligase IV-XRCC4 and DNA-PKcs also formed complexes at the ends of DNA molecules, but DNA-PKcs did not inhibit ligation. Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4. In the presence of DNA-PK, the majority of the joining events catalyzed by DNA ligase IV-XRCC4 were intermolecular because Ku inhibited intramolecular ligation, but DNA-PKcs still stimulated intramolecular ligation. We suggest that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation.     

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.     

Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining

In V(D)J recombination, the RAG proteins bind at a pair of signal sequences adjacent to the V, D, or J coding regions and cleave the DNA, resulting in two signal ends and two hairpinned coding ends. The two coding ends are joined to form a coding joint, and the two signal ends are joined to form a signal joint; this joining is done by the nonhomologous DNA end joining (NHEJ) pathway. A recombinational alternative in which a signal end is recombined with a coding end can also occur in a small percentage of the V(D)J recombination events in murine and human cells, and these are called hybrids (or hybrid joints). Two mechanisms have been proposed for the formation of these hybrids. One mechanism is via NHEJ, after initial cutting by RAGs. The second mechanism does not rely on NHEJ, but rather invokes that the RAGs can catalyze joining of the signal to the hairpinned coding end, by using the 3'OH of the signal end as a nucleophile to attack the phosphodiester bonds of the hairpinned coding end. In the present study, we addressed the question of which type of hybrid joining occurs in a physiological environment, where standard V(D)J recombination presumably occurs and normal RAG proteins are endogenously expressed. We find that all hybrids in vivo require DNA ligase IV in human cells, which is the final component of the NHEJ pathway. Hence, hybrid joints rely on NHEJ rather than on the RAG complex for joining.     

Roles of nonhomologous DNA end joining, V(D)J recombination, and class switch recombination in chromosomal translocations

When a single double-strand break arises in the genome, nonhomologous DNA end joining (NHEJ) is a major pathway for its repair. When double-strand breaks arise at two nonhomologous sites in the genome, NHEJ also appears to be a major pathway by which the translocated ends are joined. The mechanism of NHEJ is briefly summarized, and alternative enzymes are also discussed. V(D)J recombination and class switch recombination are specialized processes designed to create double-strand DNA breaks at specific locations in the genomes of lymphoid cells. Sporadic Burkitt's lymphoma and myelomas can arise due to translocation of the c-myc gene into the Ig heavy chain locus during class switch recombination. In other lymphoid neoplasms, the RAG complex can create double-strand breaks that result in a translocation. Such RAG-generated breaks occur at very specific nucleotides that are directly adjacent to sequences that resemble canonical heptamer/nonamer sequences characteristic of normal V(D)J recombination. This occurs in some T cell leukemias and lymphomas. The RAG complex also appears capable of recognizing regions for their altered DNA structure rather than their primary sequence, and this may account for the action by RAGs at some chromosomal translocation sites, such as at the bcl-2 major breakpoint region in the follicular lymphomas that arise in B lymphocytes.     

Illegitimate recombination in Xenopus: characterization of end-joined junctions.

When linear DNAs are injected into Xenopus laevis eggs, they are converted into several different kinds of recombination products. Some molecules undergo homologous recombination by a resection-annealing mechanism; some ends are precisely ligated; and some ends are joined by illegitimate means. The homologous and illegitimate products are also generated in nuclear extracts from stage VI Xenopus oocytes. In order to gain insight into the mechanism(s) of illegitimate end joining, we amplified, cloned and sequenced a number of junctions from eggs and from oocyte extracts. The egg junctions fell into three categories: some with no homology at the join point that may have been produced by blunt-end ligation; some based on small, but significant homologies (5-10 bp); and some with matches of only 1 or 2 nucleotides at the joint. Junctions made in oocyte extracts were largely of the latter type. In the extracts, formation of illegitimate joints required the addition of all four deoxyribonucleoside triphosphates and was inhibited by aphidicolin. This indicates that this process involves DNA synthesis, and mechanisms incorporating this feature are considered. The spectrum of recombination products formed in Xenopus eggs is very reminiscent of those produced from DNA introduced into mammalian cells.     

Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining

DNA double-strand break (DSB) processing was studied in mouse testicular extracts using a defined DSB created by cleaving supercoiled pUC12 DNA at a unique site as the substrate, and analysing the processed DNA by gel electrophoresis. Our results demonstrated that enzymatic activity in the extracts promoted multimerization of DNA and suppressed its circularization. This was distinctly different from T4 DNA ligase activity in the control and therefore the process must be more complex than simple ligation. Efficiency of this end-to-end joining was ATP and Mg(2+)-dependent and was much higher with cohesive (especially with 5') than with blunt ends. On recleaving, the joining was predominantly faithful, especially for cohesive ends; but a detectable fraction of DNA had undergone end-processed joining causing junctional deletions, mostly with blunt ends. Redigestion of end-joined products from time course experiments established that the end-deleted joining occurred concurrent to the faithful joining. Junctional segments were cloned and their restriction analysis confirmed the presence of large deletions from both the sides. These results suggested the association of an end-processing activity (exonuclease/helicase + flap endonuclease) along with the end-joining ligase(s). Suppression of end-edited joining on lowering the reaction temperature to 17 degrees or 14 degrees C, despite efficient faithful joining, indicated that this enzymatic activity is retarded at low temperature. Though the efficiency and fidelity of joining were termini-dependent, the orientation of joining was random. Lack of preference for homologous ends (H:H or T:T), as well as efficient joining of heterologous DNA (pUC12/pBR322) having two different blunt termini, showed that the end joining could occur independent of extensive/terminal homology. Retention of radioactivity on end joining of (alpha-32P)dCTP end-filled cohesive termini, and lack of their junctional cleavability, apparently due to restriction site duplication, suggested direct double strand ligation. Thus it is demonstrated that mouse male germ cells possess an efficient DNA end-joining activity, involving either a major pathway of precise joining, or a minor end-deleted joining, and it seems to be achieved by a multienzymatic complex as suggested also for somatic cells by others. These results show that mammalian male germ cells that are proficient in homologous recombination utilize nonhomologous end-joining (NHEJ) mechanism for DSB processing and therefore NHEJ is a conserved, universal pathway for the vital function of DSB repair.     

Joining of nonhomologous DNA double strand breaks in vitro.

Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.     

End-joining repair of double-strand breaks in Drosophila melanogaster is largely DNA ligase IV independent

Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4-independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies.     

A physical and functional interaction between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in nonhomologous end joining

Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining. Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks. Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain. This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1. Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation. Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomology-mediated pathway of nonhomologous end joining.     

The joining of non-complementary DNA double-strand breaks by mammalian extracts.

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini. This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from blunt-end joining by the biochemical fractionation of extracts. Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.     

Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination.

Linear hepadnavirus DNA in primary hepatocyte cultures efficiently participates in intra- and intermolecular nonhomologous recombination at its ends. The products of this recombination are (i) monomeric covalently closed circular DNAs (cccDNAs) with deletions and insertions around the site of joining and (ii) oligomeric forms in which monomers are joined near the ends in random orientation. A fraction of monomeric cccDNAs can serve as intermediates in further DNA replication through at least five generations of nonhomologous recombination in a process we call illegitimate replication. We suggest that the monomeric and oligomeric linear DNAs produced by illegitimate replication may be precursors of the integrated and other high-molecular-weight hepadnaviral DNA forms seen in chronic infection.     

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases λ and μ for nonhomologous end joining in human whole-cell extracts

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5′ or 3′ overhangs, and no joining at all of partially complementary 3′ overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase λ, but was restored by addition of either polymerase λ or polymerase μ. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.