Biomedical subjects

M R Lieber

Publications and source records attributed to M R Lieber.

At least 19 recordsLinked to original sources

PubMed1999

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.

14-3-3 Proteins↗

PubMed1998

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.

Animals↗

PubMed1998

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.

DNA Repair↗

PubMed1998

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].

Amino Acid Sequence↗

PubMed1998

V(D)J recombination activity in human hematopoietic cells: correlation with developmental stage and genome stability.

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.

Animals↗

PubMed1998

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.

Aging↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Alleles↗

PubMed1998

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.

Antigens, Nuclear↗

PubMed1998

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.

Animals↗

PubMed1997

Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies.

DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.

Antigens, Nuclear↗

PubMed1997

Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells.

Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4-DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.

Animals↗

PubMed1997

Yeast DNA ligase IV mediates non-homologous DNA end joining.

The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination. Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions. Here we show that S. cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 and the previously unknown DNA ligase IV homologue DNL4. dnl4 mutants are deficient in precise and end-processed NHEJ. DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects. dnl4 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation. Thus, S. cerevisiae has two distinct and separate ligation pathways.

Carboxy-Lyases↗

PubMed1997

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction.

Animals↗

PubMed1997

The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair.

Unlike the most well-characterized prokaryotic polymerase, E. coli DNA pol l, none of the eukaryotic polymerases have their own 5' to 3' exonuclease domain for nick translation and Okazaki fragment processing. In eukaryotes, FEN-1 is an endo- and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specificity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair.

DNA Repair↗

PubMed1997

Interaction between DNA-dependent protein kinase and a novel protein, KIP.

DNA-dependent protein kinase (DNA-PKcs) is the only eukaryotic kinase activated by DNA ends. Mutation of DNA-PKcs results in murine severe combined immune deficiency in mice and radiation sensitivity. Both the immune and the radiation defects are due to a failure in double-strand break repair. Biochemical studies indicate that DNA-PKcs kinase activity is stimulated by the presence of the DNA end binding protein. Ku. Autophosphorylation of DNA-PKcs results in its inactivation. Based on these studies, DNA-PKcs is presumed to play a direct and important role in the repair of double-strand breaks, but the details of its role are quite unclear. We have done two-hybrid analysis of this entire protein to identify other proteins with which it interacts. Thus far, extensive analysis has only revealed one strong interaction that satisfies both high genetic and biochemical stringency. The interaction is with a novel human protein that has 26% amino acid identity with the phosphatase component, calcineurin B. We discuss the interaction of DNA-PKcs with this novel calcium-binding protein family member in the context of possible kinase-phosphatase regulation of DNA end joining.

Adult↗

PubMed1997

Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double-strand breaks.

A convergence of information from biochemistry, yeast and mammalian genetics, immunology, and radiation biology has permitted identification of some of the protein participants - Ku, DNA-PK, XRCC4 - and the reaction intermediates in DNA end joining, suggesting how broken chromosomal ends may be recognized and repaired in eukaryotic cells. Some components may be defective in inherited disorders.

Animals↗

PubMed1997

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus.

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-->D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.

Abelson murine leukemia virus↗