Biomedical subjects

Michael R Lieber

Publications and source records attributed to Michael R Lieber.

At least 19 recordsLinked to original sources

PubMed2026

RAG-mediated structural variation and its impact on relapse risk in acute lymphoblastic leukemia.

Relapse during treatment of B-cell acute lymphoblastic leukemia (B-ALL) is a harbinger of poor outcomes. Identifying biomarkers for subsequent relapse risk which are detectable at B-ALL diagnosis remains a priority. Off-target recombination-activating gene (RAG)-mediated structural variants (SVs) generate genomic instability that drives leukemogenesis and may underlie treatment resistance. Leveraging sequencing data in 1,496 pediatric B-ALL patients enriched for relapse status (relapse n=532; non-relapse n=964), we characterized RAG-mediated SVs across B-ALL molecular subtypes and examined their association with patient characteristics and their impact on clinical outcomes. Off-target RAG-mediated SVs were overall frequent, particularly in ETV6::RUNX1, ETV6::RUNX1-like, and Ph-like B-ALL subtypes, while increasing age-at-diagnosis was positively associated with burden of off-target RAG-mediated SVs (P<.001). Off-target RAG-mediated SVs with a recombination signal sequence (RSS) at one breakpoint, a hallmark of off-target RAG activity, were significantly more frequent at diagnosis in patients who subsequently relapsed (P=.001). This association remained significant in multivariable regression analysis (per SV odds ratio [OR]:1.08, 95%CI:1.04-1.12), in minimal residual disease (MRD)-negative patients (OR:1.09, 95%CI:1.04-1.14) and across subtypes. Excluding deletions, MRD-negative ETV6::RUNX1 patients with ≥3 off-target RAG-mediated SVs had a >3-fold risk of relapse (hazard ratio:3.47, 95% CI:1.86-6.49). RAG-mediated SVs were also associated with relapse risk in T-cell ALL patients. Off-target RAG-mediated SV burden at diagnosis is a risk factor of relapse in pediatric ALL across molecular subtypes and independent of MRD status.

Journal Article↗

PubMed2006

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.

Animals↗

PubMed2006

Roles of nonhomologous DNA end joining, V(D)J recombination, and class switch recombination in chromosomal translocations.

When a single double-strand break arises in the genome, nonhomologous DNA end joining (NHEJ) is a major pathway for its repair. When double-strand breaks arise at two nonhomologous sites in the genome, NHEJ also appears to be a major pathway by which the translocated ends are joined. The mechanism of NHEJ is briefly summarized, and alternative enzymes are also discussed. V(D)J recombination and class switch recombination are specialized processes designed to create double-strand DNA breaks at specific locations in the genomes of lymphoid cells. Sporadic Burkitt's lymphoma and myelomas can arise due to translocation of the c-myc gene into the Ig heavy chain locus during class switch recombination. In other lymphoid neoplasms, the RAG complex can create double-strand breaks that result in a translocation. Such RAG-generated breaks occur at very specific nucleotides that are directly adjacent to sequences that resemble canonical heptamer/nonamer sequences characteristic of normal V(D)J recombination. This occurs in some T cell leukemias and lymphomas. The RAG complex also appears capable of recognizing regions for their altered DNA structure rather than their primary sequence, and this may account for the action by RAGs at some chromosomal translocation sites, such as at the bcl-2 major breakpoint region in the follicular lymphomas that arise in B lymphocytes.

Base Sequence↗

PubMed2006

Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination.

R-loops form at Sgamma3 and Sgamma2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine Sgamma3 and Sgamma2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the Sgamma3 and the Sgamma2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.

Animals↗

PubMed2006

DNA structures at chromosomal translocation sites.

It has been unclear why certain defined DNA regions are consistently sites of chromosomal translocations. Some of these are simply sequences of recognition by endogenous recombination enzymes, but most are not. Recent progress indicates that some of the most common fragile sites in human neoplasm assume non-B DNA structures, namely deviations from the Watson-Crick helix. Because of the single strandedness within these non-B structures, they are vulnerable to structure-specific nucleases. Here we summarize these findings and integrate them with other recent data for non-B structures at sites of consistent constitutional chromosomal translocations.

Chromosome Fragility↗

PubMed2006

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV.

DNA double-strand breaks (dsb) during V(D)J recombination of T and B lymphocyte receptor genes are resolved by the non-homologous DNA end joining pathway (NHEJ) including at least six factors: Ku70, Ku80, DNA-PK(cs), Artemis, Xrcc4, and DNA ligase IV (Lig4). Artemis and Lig4 are the only known V(D)J/NHEJ factors found deficient in human genetic disorders. Null mutations of the Artemis gene result in a complete absence of T and B lymphocytes and increased cellular sensitivity to ionizing radiations, causing radiosensitive-SCID. Mutations of Lig4 are exclusively hypomorphic and have only been described in six patients, four exhibiting mild immunodeficiency associated with microcephaly and developmental delay, while two patient had leukemia. Here we report a SCID associated with microcephaly caused by compound heterozygous hypomorphic mutations in Lig4. Residual activity of Lig4 in these patients is underscored by a normal pattern of TCR-alpha and -beta junctions in the T cells of the patients and a moderate impairment of V(D)J recombination as tested in vitro. These observations contrast with the severity of the clinical immunodeficiency, suggesting that Lig4 may have additional critical roles in lymphocyte survival beyond V(D)J recombination.

Base Sequence↗

PubMed2006

The polymerases for V(D)J recombination.

DNA transactions of a wide variety generally require three major types of enzymatic activities: nucleases, polymerases, and ligases. V(D)J recombination is no exception. In this issue, Bertocci et al. (2006) have provided new insight by generating mice deficient in one or more of the polymerases.

Animals↗

PubMed2006

Analysis of non-B DNA structure at chromosomal sites in the mammalian genome.

Changes at sites of genetic instability ultimately involve DNA repair pathways. Some sites of genetic instability in the mammalian genome appear to be unstable because they adopt a non-B DNA conformation. We describe two structural approaches for determination of whether a genomic region is configured in a non-B DNA conformation. Our studies indicate that at least some chromosomal fragile sites can be explained by such altered DNA conformations. One of the methods that we describe is called the bisulfite modification assay. This is a powerful assay because it provides information on individual DNA molecules. The second approach uses preexisting DNA structural reagents, but describes our specific application of them to analysis of DNA in vivo.

Animals↗

PubMed2006

Detection and structural analysis of R-loops.

R-loops are structures where an RNA strand is base paired with one DNA strand of a DNA duplex, leaving the displaced DNA strand single-stranded. Stable R-loops exist in vivo at prokaryotic origins of replication, the mitochondrial origin of replication, and mammalian immunoglobulin (Ig) class switch regions in activated B lymphocytes. All of these R-loops arise upon generation of a G-rich RNA strand by an RNA polymerase upon transcription of a C-rich DNA template strand. These R-loops are of significant length. For example, the R-loop at the col E1 origin of replication appears to be about 140 bp. Our own lab has focused on class switch regions, where the R-loops can extend well over a kilobase in length. Here, methods are described for detection and analysis of R-loops in vitro and in vivo.

Animals↗

PubMed2006

In vitro nonhomologous DNA end joining system.

The nonhomologous end joining (NHEJ) pathway is the major pathway that repairs DNA double strand breaks in multicellular eukaryotic organisms. Unlike homologous recombination, the NHEJ pathway utilizes minimal or no homology between the ends that need to be joined. Although the resulting NHEJ-repaired junctions can be diverse in sequence, they share a few common features, including frequent nucleolytic resection of the ends, near-random junctional additions, and utilization of microhomology. The in vitro NHEJ assay was developed in an attempt to recapitulate the joining of incompatible ends with purified core proteins and some additional factors. This in vitro system allows further understanding of the biochemical features of the pathway and evaluation of the functions of other proteins in NHEJ.

Animals↗

PubMed2005

Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining.

In V(D)J recombination, the RAG proteins bind at a pair of signal sequences adjacent to the V, D, or J coding regions and cleave the DNA, resulting in two signal ends and two hairpinned coding ends. The two coding ends are joined to form a coding joint, and the two signal ends are joined to form a signal joint; this joining is done by the nonhomologous DNA end joining (NHEJ) pathway. A recombinational alternative in which a signal end is recombined with a coding end can also occur in a small percentage of the V(D)J recombination events in murine and human cells, and these are called hybrids (or hybrid joints). Two mechanisms have been proposed for the formation of these hybrids. One mechanism is via NHEJ, after initial cutting by RAGs. The second mechanism does not rely on NHEJ, but rather invokes that the RAGs can catalyze joining of the signal to the hairpinned coding end, by using the 3'OH of the signal end as a nucleophile to attack the phosphodiester bonds of the hairpinned coding end. In the present study, we addressed the question of which type of hybrid joining occurs in a physiological environment, where standard V(D)J recombination presumably occurs and normal RAG proteins are endogenously expressed. We find that all hybrids in vivo require DNA ligase IV in human cells, which is the final component of the NHEJ pathway. Hence, hybrid joints rely on NHEJ rather than on the RAG complex for joining.

Animals↗

PubMed2005

Repair of double-strand DNA breaks by the human nonhomologous DNA end joining pathway: the iterative processing model.

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. Non-homologous 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, polymerization, 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.

Animals↗

PubMed2005

The DNA-dependent protein kinase catalytic subunit phosphorylation sites in human Artemis.

Artemis protein has irreplaceable functions in V(D)J recombination and nonhomologous end joining (NHEJ) as a hairpin and 5' and 3' overhang endonuclease. The kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is necessary in activating Artemis as an endonuclease. Here we report that three basal phosphorylation sites and 11 DNA-PKcs phosphorylation sites within the mammalian Artemis are all located in the C-terminal domain. All but one of these phosphorylation sites deviate from the SQ or TQ motif of DNA-PKcs that was predicted previously from in vitro phosphorylation studies. Phosphatase-treated mammalian Artemis and Artemis that is mutated at the three basal phosphorylation sites still retain DNA-PKcs-dependent endonucleolytic activities, indicating that basal phosphorylation is not required for the activation. In vivo studies of Artemis lacking the C-terminal domain have been reported to be sufficient to complement V(D)J recombination in Artemis null cells. Therefore, the C-terminal domain may have a negative regulatory effect on the Artemis endonucleolytic activities, and phosphorylation by DNA-PKcs in the C-terminal domain may relieve this inhibition.

Baculoviridae↗

PubMed2005

The Artemis:DNA-PKcs endonuclease cleaves DNA loops, flaps, and gaps.

In eukaryotic cells, nonhomologous DNA end joining (NHEJ) is a major pathway for repair of double-strand DNA breaks (DSBs). Artemis and the 469kDa DNA-dependent protein kinase (DNA-PKcs) together form a key nuclease for NHEJ in vertebrate organisms. The structure-specific endonucleolytic activity of Artemis is activated by binding to and phosphorylation by DNA-PKcs. We tested various DNA structures in order to understand the range of structural features that are recognized by the Artemis:DNA-PKcs complex. We find that all tested substrates that contain single-to-double-strand transitions can be cleaved by the Artemis:DNA-PKcs complex near the transition region. The cleaved substrates include heterologous loops, stem-loops, flaps, and gapped substrates. Such versatile activity on single-/double-strand transition regions is important in understanding how reconstituted NHEJ systems that lack DNA polymerases can join incompatible DNA ends and yet preserve 3' overhangs. Additionally, the flexibility of the Artemis:DNA-PKcs nuclease may be important in removing secondary structures that hinder processing of DNA ends during NHEJ.

DNA↗

PubMed2005

Evidence for a triplex DNA conformation at the bcl-2 major breakpoint region of the t(14;18) translocation.

The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.

Antibodies, Monoclonal↗

PubMed2005

Omenn syndrome due to ARTEMIS mutations.

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 alpha/beta 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.

Codon, Initiator↗

PubMed2005

Generation and characterization of endonuclease G null mice.

Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single- and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.

Animals↗

PubMed2005

Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro.

The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

Animals↗