DNA length-dependent cooperative interactions in the binding of Ku to DNA.

Despite its central role in the nonhomologous DNA end joining process, we still have an incomplete picture of the interaction between Ku and DNA. Here we describe both kinetic (surface plasmon resonance or SPR) and equilibrium (electrophoretic mobility shift assay or EMSA) studies of Ku binding to linear double-stranded DNA. Ku interaction with 1-site DNA is noncooperative, as expected. Electrophoretic mobility shift assays indicate cooperativity in the binding of Ku molecules to DNA long enough for two Ku molecules to bind (2-site DNA). For the kinetic studies, we use surface plasmon resonance in which one end of the DNA molecules is linked to a surface while the other end is free to interact with Ku. We find that one Ku molecule dissociates from 1-site DNA with simple Langmuir (i.e., independent) kinetics. However, two Ku molecules associate and dissociate from 2-site DNA with a time course that cannot be described as a simple Langmuir interaction. On 3- and 4-site DNA, EMSA and SPR studies do not reveal any cooperativity, suggesting that the middle Ku does not exhibit cooperative interaction with the two Ku molecules bound at the DNA ends. These results indicate that Ku molecules can demonstrate cooperative interaction, and this is influenced by their positions along the DNA.

Analysis of the V(D)J recombination efficiency at lymphoid chromosomal translocation breakpoints.

Chromosomal translocations and deletions are among the major events that initiate neoplasia. For lymphoid chromosomal translocations, misrecognition by the RAG (recombination activating gene) complex of V(D)J recombination is one contributing factor that has long been proposed. The chromosomal translocations involving LMO2 (t(11;14)(p13;q11)), Ttg-1 (t(11;14)(p15;q11)), and Hox11 (t(10;14)(q24;q11)) are among the clearest examples in which it appears that a D or J segment has synapsed with an adventitious heptamer/nonamer at a gene outside of one of the antigen receptor loci. The interstitial deletion at 1p32 involving SIL (SCL-interrupting locus)/SCL (stem cell leukemia) is a case involving two non-V(D)J sites that have been suggested to be V(D)J recombination mistakes. Here we have used our human extrachromosomal substrate assay to formally test the hypothesis that these regions are V(D)J recombination misrecognition sites and, more importantly, to quantify their efficiency as V(D)J recombination targets within the cell. We find that the LMO2 fragile site functions as a 12-signal at an efficiency that is only 27-fold lower than that of a consensus 12-signal. The Ttg-1 site functions as a 23-signal at an efficiency 530-fold lower than that of a consensus 23-signal. Hox11 failed to undergo recombination as a 12- or 23-signal and was at least 20,000-fold less efficient than consensus signals. SIL has been predicted to function as a 12-signal and SCL as a 23-signal. However, we find that SIL actually functions as a 23-signal. These results provide a formal demonstration that certain chromosomal fragile sites can serve as RAG complex targets, and they determine whether these sites function as 12- versus 23-signals. These results quantify one of the three major factors that determine the frequency of these translocations in T-cell acute lymphocytic leukemia.

The nicking step in V(D)J recombination is independent of synapsis: implications for the immune repertoire.

In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking. Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.

Stable RNA/DNA hybrids in the mammalian genome: inducible intermediates in immunoglobulin class switch recombination.

Although it is well established that mammalian class switch recombination is responsible for altering the class of immunoglobulins, the mechanistic details of the process have remained unclear. Here, we show that stable RNA/DNA hybrids form at class switch sequences in the mouse genome upon cytokine-specific stimulation of class switch in primary splenic B cells. The RNA hybridized to the switch DNA is transcribed in the physiological orientation. Mice that constitutively express an Escherichia coli ribonuclease H transgene show a marked reduction in RNA/DNA hybrid formation, an impaired ability to generate serum immunoglobulin G antibodies, and significant inhibition of class switch recombination in their splenic B cells. These data provide evidence that stable RNA/DNA hybrids exist in the mammalian nuclear genome, can serve as intermediates for physiologic processes, and are mechanistically important for efficient class switching in vivo.

Transcription-dependent R-loop formation at mammalian class switch sequences.

Immunoglobulin class switching is mediated by recombination between switch sequences located immediately upstream of the immunoglobulin constant heavy chain genes. Targeting of recombination to particular switch sequences is associated temporally with transcription through these regions. We recently have provided evidence for inducible and stable RNA-DNA hybrid formation at switch sequences in the mouse genome that are mechanistically important for class switching in vivo. Here, we define in vitro the precise configuration of the DNA and RNA strands within this hybrid structure at the Smicro, Sgamma3 and Sgamma2b mouse switch sequences. We find that the G-rich (non-template) DNA strand of each switch sequence is hypersensitive to probes throughout much of its length, while the C-rich (template) DNA strand is essentially resistant. These results demonstrate formation of an R-loop, whereby the G-rich RNA strand forms a stable heteroduplex with its C-rich DNA strand counterpart, and the G-rich DNA strand exists primarily in a single-stranded state. We propose that the organized structure of the R-loop is essential for targeting the class switch recombination machinery to these sequences.

The mammalian FEN-1 locus: structure and conserved sequence features.

Flap endonuclease 1 (FEN-1) is an enzyme that is very important for DNA replication in all eukaryotes because it cleaves the 5' DNA flaps that arise between Okazaki fragments. In addition, FEN-1 is important for base excision repair and for nonhomologous DNA end joining in all eukaryotes from yeast to human. Here we report the structure and sequence of the murine genomic FEN-1 locus, and we compare it to the human FEN-1 locus. The transcriptional initiation zone of FEN-1 is within a CpG island, and the coding region of FEN-1 is a single exon in both the murine and human genomes. There are striking regions of nucleotide sequence homology within the 5' or 3'UTR or immediately upstream of the 5'UTR. These regions range from 30 to 230 bp. The functions of these conserved sequence blocks could be in transcriptional regulation, or they may represent a gene that overlaps in its initiation zone with FEN-1, but is oriented in the opposite transcriptional direction.

The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts.

There are two types of chromosome instability, structural and numerical, and these are important in cancer. Many structural abnormalities are likely to involve double-strand DNA (dsDNA) breaks. Nonhomologous DNA end joining (NHEJ) and homologous recombination are the major pathways for repairing dsDNA breaks. NHEJ is the primary pathway for repairing dsDNA breaks throughout the G0, G1 and early S phases of the cell cycle [1]. Ku86 and DNA ligase IV are two major proteins in the NHEJ pathway. We examined primary dermal fibroblasts from mice (wild type, Ku86(+/-), Ku86(-/-), and DNA ligase IV(+/-)) for chromosome breaks. Fibroblasts from Ku86(+/-) or DNA ligase IV(+/-) mice have elevated frequencies of chromosome breaks compared with those from wild-type mice. Fibroblasts from Ku86(-/-) mice have even higher levels of chromosome breaks. Primary pre-B cells from the same animals did not show significant accumulation of chromosome breaks. Rather the pre-B cells showed increased cell death. These studies demonstrate that chromosome breaks arise frequently and that NHEJ is required to repair this constant spontaneous damage.

Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination.

V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some 12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved.

Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway.

Repair of DNA double strand breaks by nonhomologous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible. We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase beta) in repair at a variety of end configurations. Mutation of POL4 did not impair DNA polymerase-independent religation of fully compatible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymerization. In contrast, the frequency of joins that also required removal of a 5'- or 3'-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1, or rad1) yeast. In a chromosomal double strand break assay, pol4 mutation conferred a marked increase in sensitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3'-terminal mismatch. The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus. Paradoxically, in vitro analyses with oligonucleotide substrates demonstrated that although Pol4 fills gaps with displacement of mismatched but not matched 5' termini, it lacks both 5'- and 3'-terminal nuclease activities. Pol4 is thus specifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases.

The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes.

Recent progress over the past year has provided new insights into the proteins involved in nonhomologous end joining. The assembly of Ku and DNA-dependent protein kinase at DNA ends is now understood in greater detail. Murine genetic knockouts for DNA ligase IV and XRCC4 are embryonic lethal, indicating that nonhomologous end joining is essential for viability. Interestingly, neurones, in addition to lymphocytes, are particularly vulnerable to an absence of NHEJ.

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 ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes.

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.

Warner-Lambert/Parke-Davis Award Lecture. Pathological and physiological double-strand breaks: roles in cancer, aging, and the immune system.

Pathological agents such as ionizing radiation and oxidative free radicals can cause breaks in both strands of the DNA at a given site (double-strand break). This is the most serious type of DNA damage because neither strand is able to provide physical integrity or information content, as would be the case for single-strand DNA damage where one strand of the duplex remains intact. The repair of such breaks usually results in an irreversible alteration of the DNA. Two physiological forms of intentional double-strand (ds) DNA breakage and rejoining occur during lymphoid differentiation. One is V(D)J recombination occurring during early B and T cell development, and the other is class switch recombination, occurring exclusively in mature B cells. The manner in which physiological and most pathological double-strand DNA breaks are rejoined to restore chromosomal integrity are the same. Defects during the phases in which pathological or physiological breaks are generated or in which they are joined can result in chromosomal translocations or loss of genetic information at the site of breakage. Such events are the first step in some cancers and may be a key contributor to changes in DNA with age. Inherited defects in this process can result in severe combined immune deficiency. Hence, pathological and physiological DNA double-strand breaks are related to immune defects and cancer and may be one of the key ways in which DNA is damaged during aging.

The RAG-HMG1 complex enforces the 12/23 rule of V(D)J recombination specifically at the double-hairpin formation step.

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12x12 and 23x23 substrates as well as 12x23 substrates. However, hairpin formation occurs at both of the signals only on 12x23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.

Productive and nonproductive complexes of Ku and DNA-dependent protein kinase at DNA termini.

DNA-dependent protein kinase (DNA-PK) is the only eukaryotic protein kinase known to be specifically activated by double-stranded DNA (dsDNA) termini, accounting for its importance in repair of dsDNA breaks and its role in physiologic processes involving dsDNA breaks, such as V(D)J recombination. In this study we conducted kinase and binding analyses using DNA-PK on DNA termini of various lengths in the presence and absence of Ku. We confirmed our previous observations that DNA-PK can bind DNA termini in the absence of Ku, and we determined rate constants for binding. However, in the presence of Ku, DNA-PK can assume either a productive or a nonproductive configuration, depending on the length of the DNA terminus. For dsDNA greater than 26 bp, the productive mode is achieved and Ku increases the affinity of the DNA-PK for the Ku:DNA complex. The change in affinity is achieved by increases in both the kinetic association rate and reduction in the kinetic dissociation rate. For dsDNA smaller than 26 bp, the nonproductive mode, in which DNA-PK is bound to Ku:DNA but is inactive as a kinase, is assumed. Both the productive and nonproductive configurations are likely to be of physiologic importance, depending on the distance of the dsDNA break site to other protein complexes, such as nucleosomes.

Requirement for an interaction of XRCC4 with DNA ligase IV for wild-type V(D)J recombination and DNA double-strand break repair in vivo.

The XRCC4 gene is required for the repair of DNA double-strand breaks in mammalian cells. Without XRCC4, cells are hypersensitive to ionizing radiation and deficient for V(D)J recombination. It has been demonstrated that XRCC4 binds and stimulates DNA ligase IV, which has led to the hypothesis that DNA ligase IV is essential for both of these processes. In this study deletion mutants of XRCC4 were tested for their ability to associate with DNA ligase IV in vitro and for their ability to reconstitute XRCC4-deficient cells in vivo. We find that a central region of XRCC4 from amino acids 100-250 is necessary for DNA ligase IV binding and that deletions within this region functionally inactivates XRCC4. Deletions within the C-terminal 84 amino acids neither affect DNA ligase IV binding nor the in vivo function of XRCC4. The correlation between the ability or inability of XRCC4 to bind DNA ligase IV and its ability or failure to reconstitute wild-type DNA repair in vivo, respectively, demonstrates for the first time that the physical interaction with DNA ligase IV is crucial for the in vivo function of XRCC4. Deletions within the N-terminal 100 amino acids inactivate XRCC4 in vivo but leave DNA ligase IV binding unaffected. This indicates further DNA ligase IV-independent functions of XRCC4.

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].

DNA-PK is essential only for coding joint formation in V(D)J recombination.

The analysis of the role of DNA-dependent protein kinase (DNA-PK) in DNA double-strand break repair and V(D)J recombination is based primarily on studies of murine scid, in which only the C-terminal 2% of the protein is deleted and the remaining 98% is expressed at levels that are within an order of magnitude of normal. In murine scid, signal joint formation is observed at normal levels, even though coding joint formation is reduced over three orders of magnitude. In contrast, a closely associated protein, Ku, is necessary for both coding and signal joint formation. Based on these observations, a reasonable hypothesis has been that absence of the DNA-PK protein (rather than merely its C-terminal 2% truncation) would ablate signal joint formation along with coding joint formation. In fact, a study of equine SCID, in which there is a much larger truncation of the DNA-PK protein, has suggested that signal joints do fail to form. In our current study, we have analyzed signal and coding joint formation in a malignant glioma cell line, M059J, which was previously shown to be deficient in DNA-PK. Our quantitative analysis shows that full-length protein levels are reduced at least 200-fold, to a level that is undetectable, yet signal joint formation occurs at wild-type levels. This result demonstrates that at least this form of non-homologous DNA end joining can occur in the absence of DNA-PK.

Antigen receptor gene rearrangement.

Two specialized forms of site-directed double-strand (ds) DNA breakage and rejoining are part of the physiologic program of lymphocytes. One is recombination of the V, D and J gene sequences, termed V(D)J recombination, occurring during early B- and T-cell development, and the other is class-switch recombination occurring exclusively in mature B cells. For V(D)J recombination significant progress has been made recently elucidating the biochemistry of the reaction. In particular our understanding of how DNA ds breaks are both generated and rejoined has increased. For class-switch recombination no definitive information is known about the nucleases required for making the ds breaks, but recent evidence suggests that the joining phase shares activities also required for V(D)J recombination and general DNA ds break repair.