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New work on DNA polymerase λ highlights its remarkable flexibility. This fits with the generally adaptable nature of the DNA-repair process in which this enzyme is involved—nonhomologous end-joining—which allows this mechanism to handle diverse types of broken DNA ends in order to restore the duplex structure, albeit with a loss of information at the join.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Analysis of chromosomal translocation sequence locations in human lymphomas has provided valuable clues about the mechanism of the translocations and when they occur. Biochemical analyses on the mechanisms of DNA breakage and rejoining permit formulation of detailed models of the human chromosomal translocation process in lymphoid neoplasms. Most human lymphomas are derived from B cells in which a DNA break at an oncogene is initiated by activation-induced deaminase (AID). The partner locus in many cases is located at one of the antigen receptor loci, and this break is generated by the recombination activating gene (RAG) complex or by AID. After breakage, the joining process typically occurs by non-homologous DNA end-joining (NHEJ). Some of the insights into this mechanism also apply to translocations that occur in non-lymphoid neoplasms.

The interplay of transcription, topological tension, and chromosome breakage is a subject of intense interest, but, with so many facets to the problem, it is difficult to test. Here, we vary the orientation of promoters relative to one another in a yeast system that permits sensitive detection of chromosome breaks. Interestingly, convergent transcription that would direct RNA polymerases into one another does not increase chromosome breakage. In contrast, divergent transcription that would create underwound and potentially single-stranded DNA does cause a marked increase in chromosome breakage. Furthermore, we examine the role that topoisomerases are playing in preventing genome instability at these promoters and find that Top2 is required to prevent instability at converging promoters.

In a recent paper published in Cell Research, a cryo-EM structure reveals the interface between DNA-PKcs and the Ku70/80:DNA complex, together forming the DNA-dependent protein kinase holoenzyme in non-homologous DNA end joining. Insight from this structure suggests how an allosteric rearrangement of DNA-PKcs driven by Ku70/80:DNA binding regulates kinase activity in this largest member of a family of structurally homologous phosphoinositide 3-kinase-related protein kinases that includes mTOR, ATR, and ATM.

R loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R loop formation, and on the number of WGCW sites.

In vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig–BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig–BCL6 and non-Ig–BCL6 rearrangements.

BCL6translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6translocation breakpoints clustered in a 2156-bp segment of BCL6intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6breaks in Ig–BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig–BCL6 and non-Ig–BCL6 rearrangements.

Previous studies have implicated activation-induced cytidine deaminase (AID) in B-cell translocations but have failed to identify any association between their chromosomal breakpoints and known AID target sequences. Analysis of 56 unclustered IgH-CCND1 translocations in mantle cell lymphoma across the ∼ 344-kb bcl-1 breakpoint locus demonstrates that half of the CCND1 breaks are near CpG dinucleotides. Most of these CpG breaks are at CGC motifs, and half of the remaining breaks are near WGCW, both known AID targets. These findings provide the strongest evidence to date that AID initiates chromosomal breaks in translocations that occur in human bone marrow B-cell progenitors. We also identify WGCW breaks at the MYC locus in Burkitt lymphoma translocations and murine IgH-MYC translocations, both of which arise in mature germinal center B cells. Finally, we propose a developmental model to explain the transition from CpG breaks in early human B-cell progenitors to WGCW breaks in later stage B cells.

Naturally-occurring ionizing radiation and reactive oxygen species (ROS) from oxidative metabolism are factors that have challenged all life forms during the course of evolution. Ionizing radiation (IR) and reactive oxygen species cause a diverse set of double-strand DNA end configurations. Nonhomologous DNA end joining (NHEJ) is an optimal DNA repair pathway for dealing with such a diverse set of DNA lesions. NHEJ can carry out nucleolytic, polymerase, and ligation operations on each strand independently. This iterative processing nature of NHEJ is ideal for repair of pathologic and physiologic double-strand breaks because it permits sequential action of the NHEJ enzymes on each DNA end and on each strand. The versatility of the Artemis:DNA-PKcs endonuclease in cleaving 5’ and 3’ overhangs, hairpins, gaps, flaps, and various loop conformations makes it well-suited for DNA end modifications on oxidized overhangs. In addition, the ability to cleave stem-loop and hairpin structures permits it to open terminal fold-back configurations that may arise at DNA ends after IR damage. The ability of the XRCC4:DNA ligase IV complex to ligate one strand without ligation of the other permits additional end joining flexibility in NHEJ and raises the possibility of optional involvement of repair proteins from other pathways.

Omenn syndrome (OS) is characterized by severe combined immunodeficiency (SCID) associated with erythrodermia, hepatosplenomegaly, lymphadenopathy, and alopecia. In patients with OS, B cells are mostly absent, T-cell counts are normal to elevated, and T cells are frequently activated and express a restricted T-cell receptor (TCR) repertoire. Thus far, inherited hypomorphic mutations of the recombination activating genes 1 and 2 (RAG1/2) have been described in OS. We report on a first patient with clinical and immunologic features of OS caused by hypomorphic ARTEMIS mutations. The patient's T cells expressed α/β receptors with an oligoclonal repertoire but normal V(D)J recombination coding joints. Sequencing of the ARTEMIS gene revealed a compound heterozygosity in this nonhomologous end-joining (NHEJ) factor, explaining the enhanced radiosensitivity of the patient's primary dermal fibroblasts. The maternal allele contained a null mutation within the active center, whereas the expression of the paternal allele with a start codon (AUG to ACG) mutation partially restored V(D)J recombination and ARTEMIS function in vivo and in vitro.

The mechanisms of chromosomal translocations in mammalian cells have been largely undefined. Recent progress on the most common translocation in human cancer, t(14;18), highlights interesting issues in DNA structure and in the enzymes involved in the cutting and joining phases of the process.

To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2B loci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, and CDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.

Flap endonuclease 1 (FEN-1) is a nuclear enzyme involved in DNA metabolism, such as replication, repair, and recombination. Here, we report the comparative genomic organization of the chicken, mouse, and human FEN1 genes as well as the comparative organization of a small gene (C11orf10) located immediately upstream of the FEN1 gene in reverse orientation. Immunostaining revealed that the C11orf10 protein, unlike FEN-1, is located in the cytoplasm, suggesting that these two proteins do not form a physical complex. Importantly, in the human genome, the two mRNAs are overlapping (14 bp) in their 5′ ends. Thus, the FEN1/C11orf10 locus is a new example of two overlapping, divergent transcription units in the human genome.

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PKcs, to that end. The Ku·DNA-PKcscomplex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP6) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP6bound to DNA-PKcs(not to Ku). Here we clearly show that, in fact, IP6associates not with DNA-PKcs, but rather with Ku. Furthermore, the binding of DNA ends and IP6to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

The human immunoglobulin heavy chain locus contains 39 functional human V(H) elements. All 39 V(H) elements (with their adjacent heptamer/nonamer signal) were tested for site-specific cleavage with purified human core RAG1 and RAG2, and HMG1 proteins in a 12/23-coupled cleavage reaction. Both nicking and hairpin formation were measured. The individual V(H) cleavage efficiencies vary over nearly a 30-fold range. These measurements will be useful in considering the factors affecting the generation of the immunoglobulin and T-cell receptor repertoires in the adult humans. Interestingly, when these cleavage efficiencies are summed for each of the V(H) families, the six V(H) family efficiencies correspond closely to the observed profile of unselected V(H) family usage in the peripheral B cells of normal adult humans. This correspondence raises the possibility that the dominant factor determining V(H) element utilization within the 1-megabase human genomic V(H) array is simply the individual RAG cleavage efficiencies.

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.

Analysis of translocation breakpoints in human B cell malignancies reveals that DNA double-strand breaks at oncogenes most frequently occur at CpG sites located within 20–600 bp fragile zones and depend on activation-induced deaminase (AID). AID requires single-stranded DNA (ssDNA) to act, but it has been unclear why or how this region transiently acquires a ssDNA state. Here, we demonstrate the ssDNA state in the 23 bp E2A fragile zone using several methods, including native bisulfite DNA structural analysis in live human pre-B cells. AID deamination within the E2A fragile zone does not require but is increased upon transcription. High C-string density, nascent RNA tails, and direct DNA sequence repeats prolong the ssDNA state of the E2A fragile zone and increase AID deamination at overlapping AID hotspots that contain the CpG sites at which breaks occur in patients. These features provide key insights into lymphoid fragile zones generally.

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 β) in repair at a variety of end configurations. Mutation of POL4did 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, pol4mutation conferred a marked increase in sensitivity to HO endonuclease in arad52background, 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 vitroanalyses 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.

ABSTRACTV(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.

Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius of at least 8.4 nm the electropores resealed within 200 ms to a radius of about 0.5 nm and stayed at that radius thereafter. Video sequences clearly show that pores are induced preferentially in the cathodal hemisphere. Pores induced in the hemisphere facing the positive electrode were either (i) never greater than 0.5 nm in radius, (ii) much smaller in number if they were greater than 0.5 nm in radius, or (iii) shorter lived. Calculations indicated that an upper limit of 700 electropores were induced per membrane.

Site‐specific DNA recombination has been identified in a wide variety of biological systems. In vertebrates, however, the only identified use of this genetic device is in the immune system. Here it plays a critical role in generating a diverse repertoire of surface receptors to intercept invading microbes and parasites. The mechanism and orchestration of this reaction are intriguing and are relevant to a broad array of related biological and biomedical issues.— Lieber, M. R. Site‐specific recombination in the immune system. FASEB J.5: 2934‐2944; 1991.

Summary Therapeutic gastric irradiation has been used to reduce peptic juice secretion in patients with peptic ulcer disease. Between 1937 and 1968 a total of 2049 patients received such therapy at the University of Chicago. Three of these patients are known to have developed sarcomas in the field of radiation. Two gastric leiomyosarcomas of the stomach were diagnosed 26 and 14 years after treatment and a malignant fibrous histiocytoma of the anterior chest wall was removed six years after gastric irradiation. Of 743 peptic ulcer patients treated without irradiation and constituted as a control group for the study of therapeutic gastric radiation, none is known to have developed sarcoma. As the incidence of sarcoma in these patient groups is known only from the tumor registry of the University of Chicago, other cases of sarcoma may exist in the groups. While an increased incidence of sarcoma has not been proven to occur in patients who received therapeutic gastric irradiation for peptic ulcer disease, the possibility of such a risk should be borne in mind by physicians caring for such patients.

V(D)J recombinase activity was measured in an array of human cell lines derived from hematopoietic malignancies representing various lineages and developmental stages. The level of recombinase activity was found to vary over a 2000-fold range between different cell lines. Several myeloid cell lines were positive for V(D)J recombinase activity, providing additional insight into the relationship between myeloid and lymphoid differentiation. Despite high levels of V(D)J recombination in two human acute lymphoblastic leukemia cell lines, the cytogenetic karyotype has remained essentially constant over several years of continuous cell culture. Silencing of recombination of chromosomal and minichromosomal targets has been strongly correlated with the replication of CpG methylated DNA in murine cells. Here, in human cells, we show that human minichromosomes bearing V(D)J recombination signals are protected well over 100-fold from recombination if they are CpG methylated, providing a rational basis for the karyotypic stability in cells with high levels of V(D)J recombination activity.

The XRCC4gene 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 IVin vitroand 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 vivofunction 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 vivofunction of XRCC4. Deletions within the N-terminal 100 amino acids inactivate XRCC4 in vivobut leave DNA ligase IV binding unaffected. This indicates further DNA ligase IV-independent functions of XRCC4.

In eukaryotic cells, a 5′-flap DNA endonuclease and a double-stranded DNA 5′-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)′-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiaeRAD2, and S. cerevisiaeYKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the Fadjstrand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the Fadjstrand using double flap structures reveals that the Fadjstrand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.

ABSTRACTDNA-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.

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.

ABSTRACTA 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 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 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.

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.

Site-specific recombination reactions in higher eukaryotes are uncommon, perhaps because of the potential genomic instability that they may create. We focus this review on the issues of site-specificity, reaction fidelity and immunologic diversity in the V(D)J recombination reaction.

The 5′〉 3′-exonuclease domain of Escherichia coliDNA polymerase I is required for the completion of lagging strand DNA synthesis, and yet this domain is not present in any of the eukaryotic DNA polymerases. Recently, the gene encoding the functional and evolutionary equivalent of this 5′〉 3′-exonuclease domain has been identified. It is called FEN-1in mouse and human cells and RTH1in Saccharomyces cerevisiae. This 42-kDa enzyme is required for Okazaki fragment processing. Here we report that FEN-1 physically interacts with proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerases δ and ∈. Through protein-protein interactions, PCNA focuses FEN-1 on branched DNA substrates (flap structures) and on nicked DNA substrates, thereby stimulating its activity 10-50-fold but only if PCNA can functionally assemble as a toroidal trimer around the DNA. This interaction is important in the physical orchestration of lagging strand synthesis and may have implications for how PCNA stimulates other members of the FEN-1 nuclease family in a broad range of DNA metabolic transactions.

Background:Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear.

No relevant conflicts of interest to declare.

The etiology of multiple myeloma remains unknown, but some heritable contribution is suspected. Very few genetic risk markers have been reported in more than one study population. A nested analysis (72 cases, 144 controls) in the Nurses’ Health Study (NHS) and Health Professionals Follow-up Study (HPFS) cohorts suggested a role in multiple myeloma susceptibility for single nucleotide polymorphisms (SNP) in the genes for the insulin receptor substrate type 1 (IRS1; rs17208470, rs1801278) and the interleukin (IL)-6 receptor (IL6R; rs6684439, rs7529229, rs2228145). The NHS and HPFS cohort members are predominantly of European origin; the present analysis further explored the IRS1 and IL6R loci in persons of European descent and in African Americans in a large pooled population comprised of five U.S. population-based studies.We used unconditional logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI) assuming a log-additive inheritance model and adjusting for age, sex, and study site. We performed all analyses separately in 824 participants of European origin (259 cases, 565 controls) and 298 African Americans (114 cases, 186 controls). We also calculated a risk allele score by summing all variant alleles across the five SNPs in the ∼95% of participants with no missing genotype data, and we used additional logistic regression models to estimate a race-specific multivariable OR and 95% CI for multiple myeloma risk per variant allele carried. All statistical tests were two-tailed and assumed an α-error of 0.05.In persons of European descent, at the locus rs17208470 (IRS1) we observed a statistically significant 42% increase in multiple myeloma risk per copy of the variant allele (OR, 95% CI: 1.42, 1.02-1.98; p=0.037). In IL6R, we observed a statistically significant association with multiple myeloma at rs6684439 (OR, 95% CI: 1.27, 1.01-1.58; p=0.037) and marginally significant associations with multiple myeloma at two loci: rs7529229 (1.24, 0.99-1.55, p=0.057) and rs2228145 (1.24, 0.99-1.55, p=0.056). The risk allele score analysis (240 cases, 532 controls of European origin with no missing genotype data) showed that the risk of multiple myeloma increased significantly by 11% per variant allele carried (OR, 95% CI per risk allele: 1.11, 1.03-1.19; p=0.008). In African Americans, two loci in IL6R, rs6684439 and rs7529229, showed suggestive non-significant associations with multiple myeloma risk (rs6684439, OR, 95% CI, p-value per copy of the minor allele: 1.37, 0.94-2.00, p=0.11; rs7529229: 1.39, 0.95-2.04, p=0.089). Neither SNP in IRS1 was associated with multiple myeloma in this group. The risk allele score analysis in African Americans (109 cases, 176 controls) suggested a 17% increase in multiple myeloma risk per variant allele carried (1.17, 0.99-1.38; p=0.067).Based on a priori hypotheses, we examined the association of five previously reported SNPs in IRS1 and IL6R with multiple myeloma in a larger series of persons of European descent than previously published, and for the first time, in African Americans. Both genes belong to biologic signaling pathways with important roles in multiple myeloma pathogenesis. In participants of European origin, SNPs in IRS1 and IL6R were at least marginally associated with multiple myeloma, and the risk of multiple myeloma increased significantly per risk allele carried. We observed slightly stronger ORs for the IL6R SNPs in African Americans, but no associations were statistically significant in that group, probably due to a lack of statistical power because of small numbers for this subgroup and/or because the true causal SNPs in these genes have differential linkage disequilibrium across racial groups. The nominally significant and suggestive associations that we observed are plausible and should be further evaluated in other large multi-ethnic studies of multiple myeloma susceptibility.Anderson: celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

The etiology of multiple myeloma remains unknown, but some heritable contribution is suspected. Very few genetic risk markers have been reported in more than one study population. A nested analysis (72 cases, 144 controls) in the Nurses' Health Study (NHS) and Health Professionals Follow-up Study (HPFS) cohorts suggested a role in multiple myeloma susceptibility for single nucleotide polymorphisms (SNP) in the genes for the insulin receptor substrate type 1 (IRS1; rs17208470, rs1801278) and the interleukin (IL)-6 receptor (IL6R; rs6684439, rs7529229, rs2228145). The NHS and HPFS cohort members are predominantly of European origin; the present analysis further explored the IRS1 and IL6R loci in persons of European descent and in African Americans in a large pooled population comprised of five U.S. population-based studies.

In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow.Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2.To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients.While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia.No relevant conflicts of interest to declare.

Greisman: Signature Genomics, LLC: Patents & Royalties. Yi:Signature Genomics, LLC: Patents & Royalties.

Abstract 398

In most cases, childhood acute lymphoblastic leukemia can be retraced to a recurrent genetic lesion in utero, which establishes a pre-leukemic clone. The TEL-AML1 fusion gene, for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only <1% of these children will eventually develop leukemia. Encounter of infectious antigen in B cell typically leads to activation of the mutator enzyme AID. While AID is required for class switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of germinal center B cells, its premature activation may be deleterious. The underlying questions for this project were (1) how are B cells during their early development safeguarded from pre-mature AID expression and (2) whether pre-mature expression of AID in early B cell development is deleterious in the sense that it pre-disposes to the clonal evolution of a pre-leukemic B cell clone in the bone marrow.We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Ra suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Ra signaling involves Stat5 phosphorylation, we studied inducible Cre-mediated deletion of Stat5, which had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Ra/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Ra/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Ra surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Ra-positive Fraction C' pre-B cells to IL7Ra-negative Fraction D pre-B cells.AID is a tightly controlled mutator enzyme in mature germinal center B cells. The factors that prevent premature expression of AID during early B cell development were not known. Here, we here we report a novel, IL7Ra/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Ra/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL.No relevant conflicts of interest to declare.

Childhood acute lymphoblastic leukemia (ALL) typically arises from a pre-leukemic pre-B cell clone, which was established in utero (Greaves and Wiemels, 2003). This led to a scenario, in which the initial prenatal lesion is followed by a series of additional transforming events, which ultimately cause malignant transformation of a pre-B cell clone. For instance, the TEL-AML1 gene rearrangement defines the most frequent type of childhood ALL and is detected in ∼1% cord blood samples compared to the cumulative risk for TEL-AML1 ALL at 1:14,000. These findings support the notion that covert pre-leukemic clones are frequent but only a small minority of them develop into frank pre-B leukemia after critical secondary genetic lesions were acquired. The postnatal mechanism(s) that drive the evolution of the fetal pre-leukemic clone towards childhood ALL are not known.We have recently demonstrated that aberrant somatic hypermutation activity of AID propagates progression of CML into lymphoid blast crisis (Klemm et al., 2009) and clonal evolution of acute lymphoblastic leukemia (Gruber et al., 2010). Here we test the hypothesis that premature expression of AID in human pre-B cells promotes the acquisition of secondary genetic lesions and propagates the clonal evolution of a pre-leukemic pre-B cell towards childhood ALL.We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes. Premature hypermutation in IL7Rα-deficient pre-B cells was consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Rα suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Rα signaling involves Stat5 phosphorylation, we studied inducible deletion of both Stat5a and Stat5b in Stat5-fl/fl pre-B cells. Inducible deletion of Stat5a and Stat5b in pre-B cells had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Rα/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Rα/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Rα surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Rα-positive Fraction C’ pre-B cells to IL7Rα-negative Fraction D pre-B cells.AID is a tightly controlled mutator enzyme, which diversifies immunoglobulin genes upon antigen-encounter of germinal center B cells. The factors that prevent premature expression of AID in pre-germinal center stage B cells were not known. Here, we here we report a novel, IL7Rα/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Rα/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Enlargement of the Fraction D pool or extension of the time window during which pre-B cells are at the Fraction D stage, may increase the risk to acquire secondary genetic lesions towards the development of childhood ALL.No relevant conflicts of interest to declare.