Biomedical subjects

Michael R Lieber

Publications and source records attributed to Michael R Lieber.

At least 37 records · Page 2Linked to original sources

PubMed2005

Both V(D)J coding ends but neither signal end can recombine at the bcl-2 major breakpoint region, and the rejoining is ligase IV dependent.

The t(14;18) chromosomal translocation is the most common translocation in human cancer, and it occurs in all follicular lymphomas. The 150-bp bcl-2 major breakpoint region (Mbr) on chromosome 18 is a fragile site, because it adopts a non-B DNA conformation that can be cleaved by the RAG complex. The non-B DNA structure and the chromosomal translocation can be recapitulated on intracellular human minichromosomes where immunoglobulin 12- and 23-signals are positioned downstream of the bcl-2 Mbr. Here we show that either of the two coding ends in these V(D)J recombination reactions can recombine with either of the two broken ends of the bcl-2 Mbr but that neither signal end can recombine with the Mbr. Moreover, we show that the rejoining is fully dependent on DNA ligase IV, indicating that the rejoining phase relies on the nonhomologous DNA end-joining pathway. These results permit us to formulate a complete model for the order and types of cleavage and rejoining events in the t(14;18) translocation.

Base Sequence↗

PubMed2005

Fine-structure analysis of activation-induced deaminase accessibility to class switch region R-loops.

Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.

Animals↗

PubMed2004

A biochemically defined system for mammalian nonhomologous DNA end joining.

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.

Animals↗

PubMed2004

Genetic interactions between BLM and DNA ligase IV in human cells.

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

Adenosine Triphosphatases↗

PubMed2004

Stability and strand asymmetry in the non-B DNA structure at the bcl-2 major breakpoint region.

The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.

Base Sequence↗

PubMed2004

Chromosomal translocations and non-B DNA structures in the human genome.

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.

Animals↗

PubMed2004

Functional and biochemical dissection of the structure-specific nuclease ARTEMIS.

During V(D)J recombination, the RAG1 and RAG2 proteins form a complex and initiate the process of rearrangement by cleaving between the coding and signal segments and generating hairpins at the coding ends. Prior to ligation of the coding ends by DNA ligase IV/XRCC4, these hairpins are opened by the ARTEMIS/DNA-PKcs complex. ARTEMIS, a member of the metallo-beta-lactamase superfamily, shares several features with other family members that act on nucleic acids. ARTEMIS exhibits exonuclease and, in concert with DNA-PKcs, endonuclease activities. To characterize amino acids essential for its catalytic activities, we mutated nine evolutionary conserved histidine and aspartic acid residues within ARTEMIS. Biochemical analyses and a novel in vivo V(D)J recombination assay allowed the identification of eight mutants that were defective in both overhang endonucleolytic and hairpin-opening activities; the 5' to 3' exonuclease activity of ARTEMIS, however, was not impaired by these mutations. These results indicate that the hairpin-opening activity of ARTEMIS and/or its overhang endonucleolytic activity are necessary but its exonuclease activity is not sufficient for the process of V(D)J recombination.

Amino Acid Sequence↗

PubMed2004

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex.

The causes of spontaneous chromosomal translocations in somatic cells of biological organisms are largely unknown, although double-strand DNA breaks are required in all proposed mechanisms. The most common chromosomal abnormality in human cancer is the reciprocal translocation between chromosomes 14 and 18 (t(14;18)), which occurs in follicular lymphomas. The break at the immunoglobulin heavy-chain locus on chromosome 14 is an interruption of the normal V(D)J recombination process. But the breakage on chromosome 18, at the Bcl-2 gene, occurs within a confined 150-base-pair region (the major breakpoint region or Mbr) for reasons that have remained enigmatic. We have reproduced key features of the translocation process on an episome that propagates in human cells. The RAG complex--which is the normal enzyme for DNA cleavage at V, D or J segments--nicks the Bcl-2 Mbr in vitro and in vivo in a manner that reflects the pattern of the chromosomal translocations; however, the Mbr is not a V(D)J recombination signal. Rather the Bcl-2 Mbr assumes a non-B-form DNA structure within the chromosomes of human cells at 20-30% of alleles. Purified DNA assuming this structure contains stable regions of single-strandedness, which correspond well to the translocation regions in patients. Hence, a stable non-B-DNA structure in the human genome appears to be the basis for the fragility of the Bcl-2 Mbr, and the RAG complex is able to cleave this structure.

Animals↗

PubMed2004

Kinetic analysis of the nicking and hairpin formation steps in V(D)J recombination.

The complete cleavage phase of V(D)J recombination includes four phases: binding of the active RAG complexes to the 12- or 23-signals, nicking of the signals, synapsis of the two signals, and hairpin formation at both signals concurrently. We have done time courses for the complete cleavage phase of the V(D)J recombination reaction and quantitated the amount of active RAG enzyme. We have also formulated a kinetic model for the binding, nicking, synapsis, and hairpin formation phases. We have utilized free solution enzymatic measurements for the binding and nicking phases as we do mathematical simulations of the kinetic model. This permits iteration of rate constants for the synapsis and hairpin formation phases until the model fits the observed overall cleavage time course. This process yields a rate constant for the hairpin formation that is 0.004 min(-1), which corresponds to an average catalytic cycle time of 250 min. This value is exceedingly close to a measured value of this constant that relied on wash-out of an inhibitory cofactor. The agreement indicates that this is likely to be the rate of the hairpin step over a wide range of range of conditions and irrespective of the DNA sequence of the V, D or J coding end located adjacent to the signal. These findings indicate that, under optimal in vitro conditions, the core RAG proteins carry out nicking at a rate which is nearly 150-fold faster than hairpin formation. The physiologic implications of this and other kinetic inferences of these time courses are discussed.

Animals↗

PubMed2004

DNA damage and aging.

The hypothesis discussed here is that a major component of aging in metazoans is oxidative damage to nuclear DNA. Such a viewpoint would be consistent with the fact that all of the thus far identified premature aging syndromes in mammals involve mutations in nuclear proteins. Several of these nuclear proteins are enzymes that are related to DNA metabolism or DNA repair. Among the single- and double-stranded DNA damage repair pathways present in eukaryotes, only one pathway often fails to restore the full information content of the genome and typically would result in a deletion of a few base pairs. This pathway is called nonhomologous DNA end joining (NHEJ) and it is a major pathway for the repair of double-strand DNA breaks. Repetitive DNA content may determine the extent to which any organism can use this pathway, and therefore, may dictate a key factor in the balance between oxidation and organismal lifespan.

Aging↗

PubMed2004

Ageing, repetitive genomes and DNA damage.

The mitochondrial production of reactive oxygen species is inversely proportional to longevity in animals. A key question now is, which molecules, among those that are oxidized, affect the lifespan of the organism most significantly?

Aging↗

PubMed2003

Developmental retinal apoptosis in Ku86-/- mice.

The nonhomologous DNA end-joining pathway (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs), is essential for maintaining genomic stability. Knockout animals for components in this pathway demonstrate a distinct pattern of cell death in the developing brain. Here we demonstrate that cell death is also present in the developing retina of E14.5 Ku86-deficient mouse embryos, suggesting that the increase in cell death in the retina is associated with chromosome breaks. In the adult retina, we do not find continuing apoptosis, but interestingly, we find decreased numbers of total neuronal cells. This suggests that the increased retinal apoptosis during embryogenesis causes the reduction in cell numbers observed in the adult retina. This analysis of the retina provides the first opportunity to formally test the hypothesis that embryonic apoptosis accounts for reduced total cell numbers in adult Ku86-/- mice.

Animals↗

PubMed2003

DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine.

Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).

Amino Acid Motifs↗

PubMed2003

Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism.

Class switch recombination is the gene rearrangement process by which our B lymphocytes change from IgM production to IgG, IgA, or IgE. Unlike the well-characterized V(D)J recombination, the mechanism of class switch recombination has been largely enigmatic until very recent progress has begun to shed light on this gene rearrangement process. Progress has been made on the enzymes involved in leading to the DNA cleavage events and on identifying the unusual DNA structures that those enzymes recognize.

Animals↗

PubMed2003

Impact of DNA ligase IV on the fidelity of end joining in human cells.

A DNA ligase IV (LIG4)-null human pre-B cell line and human cell lines with hypomorphic mutations in LIG4 are significantly impaired in the frequency and fidelity of end joining using an in vivo plasmid assay. Analysis of the null line demonstrates the existence of an error-prone DNA ligase IV-independent rejoining mechanism in mammalian cells. Analysis of lines with hypomorphic mutations demonstrates that residual DNA ligase IV activity, which is sufficient to promote efficient end joining, nevertheless can result in decreased fidelity of rejoining. Thus, DNA ligase IV is an important factor influencing the fidelity of end joining in vivo. The LIG4-defective cell lines also showed impaired end joining in an in vitro assay using cell-free extracts. Elevated degradation of the terminal nucleotide was observed in a LIG4-defective line, and addition of the DNA ligase IV-XRCC4 complex restored end protection. End protection by DNA ligase IV was not dependent upon ligation. Finally, using purified proteins, we demonstrate that DNA ligase IV-XRCC4 is able to protect DNA ends from degradation by T7 exonuclease. Thus, the ability of DNA ligase IV-XRCC4 to protect DNA ends may contribute to the ability of DNA ligase IV to promote accurate rejoining in vivo.

Base Pairing↗

PubMed2003

Overexpression of Cu/Zn superoxide dismutase is lethal for mice lacking double-strand break repair.

The non-homologous DNA end joining (NHEJ) pathway is a major double-strand DNA break repair pathway in cells of multicellular eukaryotes. Ku is a heterodimeric protein consisting of Ku70 and Ku86, and it is thought to be the first component to bind to a broken double-strand DNA end. Mice lacking Ku86 show features of premature aging, live about 6-12 months, and show a characteristic loss of neurons in the central nervous system during development. Cells from mice lacking Ku have increased numbers of chromosome breaks, a significant fraction of which are caused by oxidative metabolism. Overexpression of the cytoplasmic Cu/Zn superoxide dismutase (SOD1) from a transgene is known to increase the number of chromosome breaks in primary cells (presumably by increasing reactive oxygen species). Here we show that SOD1 overexpression in a Ku86-/- mouse results in embryonic lethality. This striking effect is, however, subject to a strain-specific modifier. Genome-wide marker analysis is most consistent with the modifier being on mouse chromosome 13. Analysis of 10 markers on chromosome 13 suggests that the modifier is within the same region as a modifier of the murine amyotropic lateral sclerosis (ALS) phenotype when it is caused by overexpression of a mutant form of SOD1. Based on these results, we propose a model in which oxidative metabolism causes chromosome breaks, leading to neuronal death; and this neuronal death may account for that seen in NHEJ mutant animals and in mammals with SOD1-mediated ALS.

Animals↗

PubMed2003

Human severe combined immune deficiency and DNA repair.

Human severe combined immune deficiency (SCID) is the most serious inherited immunological deficit. Recent work has revealed defects in the predominant pathway for double-strand break repair called nonhomologous DNA end joining, or NHEJ. Progress in the biochemistry and genetics of NHEJ and of human SCID has proven to be synergistic between these two fields in a manner that covers the range from biochemical etiology to considerations about possible gene therapy for the B- SCID patients.

Animals↗

PubMed2003

R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.

The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the gamma3 or the gamma2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism

Animals↗