Oligonucleotide capture during end joining in mammalian cells.

Extra nucleotides (termed filler DNA) are found at about 10% of the junctions of the genetic rearrangements that arise by illegitimate recombination in mammalian cells. Such filler DNAs could arise by the joining of oligonucleotide fragments to broken ends prior to end joining. We tested this possibility by microinjecting mixtures of defined oligonucleotides with SV40 genomes that were linearized in the intron for T antigen, a site where incorporation of extra nucleotides does not impair viability. Using an injection ratio of 1000 oligonucleotides per DNA end, we screened viable genomes for incorporation of single-stranded and double-stranded oligonucleotides with varying degrees of complementarity to the ends of the linear SV40 molecules. Genomes from 510 independent plaques were screened by restriction digestion to identify those that had picked up a restriction site unique to the injected oligonucleotides. Double-stranded oligonucleotides that were fully complementary to the SV40 ends were readily incorporated, but uptake of the other oligonucleotides was not detected by restriction analysis. Nucleotide sequences of junctions from 12 genomes derived from co-injection of noncomplementary oligonucleotides revealed two with filler DNA, but neither could be assigned unambiguously to the injected oligonucleotides.     

Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT’s ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT’s activity at DNA ends in vivo.     

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.     

Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.

The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.     

An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA.

Terminal deoxynucleotidyl transferase (E.C.2.7.7.3.1.) from calf thymus was used to add homopolymer tails to duplex DNA with 3' protruding, even, or 3' recessive ends. A gel electrophoresis method was employed to analyze the tail length and the percent of DNA with tails. In all the tailing reactions, dA, dT, and dC tails from CoCl2-containing buffer were longer than those from MnCl2 - or MgCl2 - containing buffers, whereas dG tails from MnCl2 -containing buffer were the longest. By varying the ratio of dNTP over DNA terminus and the concentration of terminal transferase, optimal conditions were found for adding dG or dC tails of 10-25 nucleotides in length and dA and dT tails of 20-40 nucleotides in length to duplex DNA with all types of 3' termini.     

Sperm chromatin damage associated with male smoking

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.     

Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.

Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.     

Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes.

When microinjected foreign genes integrate into the genomes of mice, multiple copies are frequently found clustered together at one location. How they concatamerize--by the integration of large linearized concatamers that are formed by simple end-to-end linkage, by circularization of individual DNA fragments and recombination, or by some other means--is not understood. In the transgenic animals studied thus far by ourselves and others, integration frequency and transgene copy number do not seem to be significantly influenced by the complementarity of the ends of the DNA fragments that have been microinjected. We have utilized PCR amplification and DNA sequence analysis to study selected transgene junctions at the nucleotide level. In two transgenic mice carrying the synthetic RSVcat gene (injected with noncomplementary overhangs on the fragment ends), ends were 'nibbled' from 1 to 62 bases before being joined to an adjacent gene copy. Repeated dinucleotides, providing the most minimal of homologies, are present in half of the characterized junctions. Determination of the relative copy number of the junctions in each mouse supports the idea that transgene complexes can undergo additional rearrangements after the initial formation event.     

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.     

Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation

In situ presence of numerous DNA strand breaks is a typical feature of apoptotic cells. Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated DNA precursors has recently been proposed as a method of analysing DNA replication. Detection of DNA strand breaks, thus, enables one to identify apoptotic and/or DNA replicating cells. The current methods for DNA strand break labelling rely on the use of exogenous terminal deoxynucleotidyl transferase which either directly attaches the fluorochrome conjugated triphosphodeoxynucleotides to 3′OH ends in the breaks, or indirectly labels 3′OH ends with digoxygenin or biotin conjugated triphosphodeoxynucleotides. A limitation of these methodologies, especially restricting their routine application in the clinic, is high cost of reagents. In the present study we have tested whether relatively simple compound BrdUTP, which is approximately three orders of magnitude less expensive than dUTP conjugated to digoxygenin, can be used as marker of DNA strand breaks. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin. The incorporated BrdUTP was detected by fluoresceinated anti-BrdUrd MoAb. Cellular fluorescence was measured by flow cytometry as well as by Laser Scanning Cytometer (LSC). The data show that intensity of DNA strand break labelling with BrdUTP was nearly four- and two-fold higher than that obtained with the indirect labelling using biotin- or digoxygenin-conjugated dUTP, respectively, and over eight-fold higher than in the case of direct labelling with the fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides. The increased labelling of DNA strand breaks with BrdUTP may reflect more efficient incorporation of this precursor by terminal transferase, compared to the nucleotides with bulky fluorochrome conjugates. DNA strand break labelling with BrdUTP, thus, offers a possibility of more sensitive (and at lower cost) detection of apoptotic or DNA replicating cells, compared to the alternative methods of DNA strand break labelling.     

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in<Emphasis Type="Italic"> Streptomyces ambofaciens</Emphasis> ATCC 23877

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.     

An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.     

Mechanistic constraints on diversity in human V(D)J recombination.

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.     

Lesion bypass by human DNA polymerase mu reveals a template-dependent, sequence-independent nucleotidyl transferase activity

DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.     

Intramolecular transposition by Tn10

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.     

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.     

Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Smicro segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but find very few mutations in GL Sgamma3 and Sgamma1 regions in activated B cells. These data suggest that switch recombination initiates in the Smicro segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Smicro segments differ, suggesting different repair mechanisms. Mutations in recombined Smicro regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Smicro do not. Additionally, induction conditions affect mutation specificity within the GL Smicro segment. Mutations are most frequent near the S-S junctions and decrease rapidly with distance from the junction. Finally, we find that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S-S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities.     

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.