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.     

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.     

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 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.     

V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.     

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.     

Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Synapsis-Mediated Fusion of Free DNA Ends Forms Inverted Dimer Plasmids in Yeast

When yeast (Saccharomyces cerevisiae) is transformed with linearized plasmid DNA and the ends of the plasmid do not share homology with the yeast genome, circular inverted (head-to-head) dimer plasmids are the principal product of repair. By measurements of the DNA concentration dependence of transformation with a linearized plasmid, and by transformation with mixtures of genetically marked plasmids, we show that two plasmid molecules are required to form an inverted dimer plasmid. Several observations suggest that homologous pairing accounts for the head-to-head joining of the two plasmid molecules. First, an enhanced frequency of homologous recombination is detected when genetically marked plasmids undergo end-to-end fusion. Second, when a plasmid is linearized within an inverted repeat, such that its ends could undergo head-to-tail homologous pairing, it is repaired by intramolecular head-to-tail joining. Last, in the joining of homologous linearized plasmids of different length, a shorter molecule can acquire a longer plasmid end by homologous recombination. The formation of inverted dimer plasmids may be related to some forms of chromosomal rearrangement. These might include the fusion of broken sister chromatids in the bridge-breakage-fusion cycle and the head-to-head duplication of genomic DNA at the sites of gene amplifications.     

Sealing of gaps in duplex DNA by T4 DNA ligase.

Single-strand gaps in DNA molecules were found to be a substrate for T4 DNA ligase. Sealing of the gaps was optimal at the same conditions as ligation of blunt-ended DNA molecules. Spermidine at a concentration of 2 mM stimulated the ligation of gaps, as well as the joining of DNA molecules with cohesive and blunt ends. In addition, spermidine reduced the optimal ATP concentration. The ligation of single-stranded gaps was a slow process, reaching a plateau after several hours at 25 degrees C. Approximately 10% of circular duplex plasmid pBR322 DNA molecules with a gap of 1-5 nucleotides could be converted to a covalently closed form. When such molecules were used for transformation of E. coli cells deletion mutants were obtained at a high frequency. The size and position of the gaps and the deletions were equivalent, confirming that T4 DNA ligase was sealing the gaps.     

DNA ligase from Drosophila melanogaster embryos. Substrate specificity and mechanism of action

DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B. A., Hawley, R. S., and Chase, J. W. (1986) J. Biol. Chem. 261, 10637-10645). This enzyme had an apparent Km for ATP of 1.6 microM. Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction. The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM. The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM. Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA. In addition, p(dT)7U could be joined when annealed with poly(dA). No joining was detected when RNA served as the template. Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA. The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases. The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible. Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.     

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5′-base overhang. Regardless of the 5′-end structure, all bleomycin-induced DSBs possess 3′-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of ≤0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires ≤100 ng substrate DNA and ≥50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.     

Recognition sequence of restriction endonuclease KpnI from Klebsiella pneumoniae.

We have determined the recognition sequence of the restriction endonuclease KpnI, previously isolated from Klebsiella pneumoniae. The enzyme cleaves the twofold rotationally symmetric sequence (see book for formula) at the positions indicated by the arrows, producing 3' protruding cohesive ends, four nucleotides in length. The specific cleavage site was unambiguously deduced using both 3' and 5' end analyses of KpnI generated restriction fragments of simian-virus 40 (SV40) DNA (1 site), adenovirus-2 (Ad-2) DNA (8 sites), and a plasmid (pCRI) DNA (2 sites).     

Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein.

The avian myeloblastosis virus integration protein (IN) was capable of removing a specific set of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat (LTR) substrates which resembled linear viral DNA in vivo. The 3'-OH-recessed ends map to the in vivo site of integration on linear viral DNA. The linear DNA plasmid substrate was formed by the generation of a unique DraI restriction enzyme site (TTT/AAA) at the circle junction of a 330-bp tandem LTR-LTR insert. IN preferentially released the three T nucleotides from the minus strand of the U3 LTR substrate compared with its ability to remove the three T nucleotides from the plus strand of the U5 LTR substrate. It was also observed that IN was capable of cleaving a non-LTR DNA substrate containing sequence homology to the U5 LTR terminus.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions.

Double strand DNA breaks in plants are primarily repaired via non-homologous end joining. However, little is known about the molecular events underlying this process. We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells. A variety of sequences were found at novel end junctions. Joining with no sequence alterations was rare. In most cases, deletions were found at both ends, and rejoining usually occurred at short repeats. A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in approximately 30% of the analyzed clones. The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA. Some insertions had a complex structure consisting of several reshuffled plasmid-related regions. These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches. Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4. It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants.     

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.