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Biomedical subjects

M Gellert

Publications and source records attributed to M Gellert.

At least 19 recordsLinked to original sources
PubMed2003

Dynamo effect in a driven helical flow.

The Roberts flow, a helical flow in the form of convectionlike rolls, is known to be capable of both kinematic and nonlinear dynamo action. We study the Roberts dynamo with particular attention being paid to the spatial structure of the generated magnetic field and its back-reaction on the flow. The dynamo bifurcation is decisively determined by the symmetry group of the problem, which is given by a subgroup of discrete transformations and a continuous translational invariance of the flow. In the bifurcation the continuous symmetry is broken while the discrete subgroup symmetry completely survives. Its actions help in understanding the spatial structures of the magnetic field and of the modified flow. In accordance with experimental observations, the magnetic field component perpendicular to the originally invariant direction is much stronger than the component in this direction. Furthermore, the magnetic field is largely concentrated in layers separating the convectionlike rolls of the flow and containing, in particular, its stagnation points, which are isolated for the modified flow while they are line filling for the original Roberts flow. The magnetic field is strongest near beta-type stagnation points, with a two-dimensional unstable and a one-dimensional stable manifold, and is weak near alpha-type stagnation points, with a two-dimensional stable and a one-dimensional unstable manifold. This contrasts with the usual picture that dynamo action is promoted at the alpha points and impeded at the beta points. Both the creation of isolated stagnation points and the concentration of strong fields at the beta points may be understood as a result of the way in which the Roberts dynamo saturates. It is also found that, while the original Roberts flow is regular, the modified flow is chaotic in the layers between the convectionlike rolls where the magnetic field is concentrated. This chaoticity, which results from the back-reaction of the magnetic field on the flow, appears to merely enhance magnetic diffusion rather than to strengthen the dynamo effect.

Journal Article↗
PubMed2001

Intermediates in V(D)J recombination: a stable RAG1/2 complex sequesters cleaved RSS ends.

Rearrangement of gene segments to generate antigen receptor coding regions depends on the RAG1/2 recombinase, which assembles a synaptic complex between two DNA signal sequences and then cleaves the DNA directly adjacent to the paired signals. After coupled cleavage of complementary signal sequences, virtually all of the cleaved signal ends remained associated with RAG1/2 in stable complexes. These signal end complexes were distinct from various precleavage RAG1/2 signal complexes in that they were resistant to treatment with heparin. A mammalian joining apparatus consisting of purified Ku70/86, XRCC4, and DNA ligase IV proteins was sufficient to join deproteinized cleaved ends, but retention of signal sequences within the signal end complex blocked access to the DNA ends and prevented their joining by these proteins. Sequestration of cleaved ends within the signal end complex would account for the persistence of these ends in the cell after cleavage and may explain why they do not normally activate the DNA-damage-dependent cell cycle checkpoint.

Antigens, Nuclear↗
PubMed2001

Direct DNA binding by Brca1.

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Animals↗
PubMed2001

A Ku bridge over broken DNA.

The Ku heterodimer is essential for the nonhomologous end-joining pathway of DNA double-strand break repair; it both protects the broken ends and recruits some of the many proteins required to complete repair. The recently determined structure of Ku provides insights into how it can both bind to the DNA ends and allow access by the other proteins required to rejoin them.

Animals↗
PubMed2000

Crystal structure of the Xrcc4 DNA repair protein and implications for end joining.

XRCC4 is essential for carrying out non-homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4-defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 A resolution. Xrcc4 protein forms a strikingly elongated dumb-bell-like tetramer. Each of the N-terminal globular head domains consists of a beta-sandwich and a potentially DNA-binding helix- turn-helix motif. The C-terminal stalk comprising a single alpha-helix >120 A in length is partly incorporated into a four-helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV. The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

Amino Acid Sequence↗
PubMed2000

RAG1/2-mediated resolution of transposition intermediates: two pathways and possible consequences.

During B and T cell development, the RAG1/RAG2 protein complex cleaves DNA at conserved recombination signal sequences (RSS) to initiate V(D)J recombination. RAG1/2 has also been shown to catalyze transpositional strand transfer of RSS-containing substrates into target DNA to form branched DNA intermediates. We show that RAG1/2 can resolve these intermediates by two pathways. RAG1/2 catalyzes hairpin formation on target DNA adjacent to transposed RSS ends in a manner consistent with a model leading to chromosome translocations. Alternatively, disintegration removes transposed donor DNA from the intermediate. At high magnesium concentrations, such as are present in mammalian cells, disintegration is the favored pathway of resolution. This may explain in part why RAG1/2-mediated transposition does not occur at high frequency in cells.

B-Lymphocytes↗
PubMed2000

A mechanistic basis for Mre11-directed DNA joining at microhomologies.

Repair of DNA double-strand breaks in vertebrate cells occurs mainly by an end-joining process that often generates junctions with sequence homologies of a few nucleotides. Mre11 is critical for this mode of repair in budding yeast and has been implicated in the microhomology-based joining. Here, we show that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends. Addition of mismatched DNA ends stimulates degradation of DNA by Mre11, whereas cohesive ends strongly inhibit it. Furthermore, if a sequence identity is revealed during the course of degradation, it causes Mre11 nuclease activity to pause, thus stabilizing the junction at a site of microhomology. A nuclease-deficient Mre11 mutant that still binds DNA can also stimulate degradation by wild-type Mre11, suggesting that Mre11-DNA complexes may interact to bridge DNA ends and facilitate DNA joining.

Catalysis↗
PubMed1999

Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex.

The Nijmegen breakage syndrome gene product (Nbs1) was shown recently to associate in vivo with the Mre11 and Rad50 proteins, which play pivotal roles in eukaryotic DNA double-strand break repair, meiotic recombination, and telomere maintenance. We show in this work that the triple complex of recombinant Nbs1, Mre11, and Rad50 proteins binds cooperatively to DNA and forms a distinct protein-DNA species. The Mre11/Rad50/Nbs1 complex displays several enzymatic activities that are not seen without Nbs1, including partial unwinding of a DNA duplex and efficient cleavage of fully paired hairpins. Unwinding and hairpin cleavage are both increased by the presence of ATP. On nonhairpin DNA ends, ATP controls a switch in endonuclease specificity that allows Mre11/Rad50/Nbs1 to cleave a 3'-protruding strand at a double-/single-strand transition. Mutational analysis demonstrates that Rad50 is responsible for ATP binding by the complex, but the ATP-dependent activities are expressed only with Nbs1 present.

Adenosine Triphosphate↗
PubMed1999

Interactions of CcdB with DNA gyrase. Inactivation of Gyra, poisoning of the gyrase-DNA complex, and the antidote action of CcdA.

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.

Bacterial Proteins↗
PubMed1999

DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity.

Mammalian cells are protected from the effects of DNA double-strand breaks by end-joining repair. Cells lacking the Xrcc4 protein are hypersensitive to agents that induce DNA double-strand breaks, and are unable to complete V(D)J recombination. The residual repair of broken DNA ends in XRCC4-deficient cells requires short sequence homologies, thus possibly implicating Xrcc4 in end alignment. We show that Xrcc4 binds DNA, and prefers DNA with nicks or broken ends. Xrcc4 also binds to DNA ligase IV and enhances its joining activity. This stimulatory effect is shown to occur at the adenylation of the enzyme. DNA binding of Xrcc4 is correlated with its complementation of the V(D)J recombination defects in XRCC4-deficient cells, but is not required for stimulation of DNA ligase IV. Thus, the ability of Xrcc4 to bind to DNA suggests functions independent of DNA ligase IV.

Animals↗
PubMed1998

DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations.

The RAG1 and RAG2 proteins are known to initiate V(D)J recombination by making a double-strand break between the recombination signal sequence (RSS) and the neighboring coding DNA. We show that these proteins can also drive the coupled insertion of cleaved recombination signals into new DNA sites in a transpositional reaction. This RAG-mediated DNA transfer provides strong evidence for the evolution of the V(D)J recombination system from an ancient mobile DNA element and suggests that repeated transposition may have promoted the expansion of the antigen receptor loci. The inappropriate diversion of V(D)J rearrangement to a transpositional pathway may also help to explain certain types of DNA translocation associated with lymphatic tumors.

DNA↗
PubMed1998

Rejoining of DNA by the RAG1 and RAG2 proteins.

Assembly of immunoglobulin and T cell receptor genes from separate gene segments [V(D)J recombination] begins with DNA double-strand breakage by the RAG1 and RAG2 proteins, acting at a pair of recombination signal sequences (RSSs). Here, the RAG proteins are shown to reverse the cleavage reaction by joining an RSS to a broken coding sequence end. These "hybrid joints" have also been found in lymphoid cells, even when the normal pathway of DNA double-strand break repair is inactive, and can now be explained by this activity of the RAG proteins.

DNA↗
PubMed1998

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks.

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.

Antigens, Nuclear↗
PubMed1998

The 3' to 5' exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks.

MRE11 and RAD50 are known to be required for nonhomologous joining of DNA ends in vivo. We have investigated the enzymatic activities of the purified proteins and found that Mre11 by itself has 3' to 5' exonuclease activity that is increased when Mre11 is in a complex with Rad50. Mre11 also exhibits endonuclease activity, as shown by the asymmetric opening of DNA hairpin loops. In conjunction with a DNA ligase, Mre11 promotes the joining of noncomplementary ends in vitro by utilizing short homologies near the ends of the DNA fragments. Sequence identities of 1-5 base pairs are present at all of these junctions, and their diversity is consistent with the products of nonhomologous end-joining observed in vivo.

Amino Acid Sequence↗
PubMed1998

Assembly of a 12/23 paired signal complex: a critical control point in V(D)J recombination.

The 12/23 rule requires that V(D)J recombination only occurs between recombination signals with 12 and 23 base pair spacers. We show that the 12/23 rule is established prior to DNA cleavage, by the formation of a synaptic complex containing both 12-spacer and 23-spacer signals. The RAG1 and RAG2 proteins, as well as the DNA bending protein HMG1, are needed for efficient formation of this complex. We show further that the synaptic complex is the functional complex for coupled cleavage. After cleavage, all four broken DNA ends remain associated with the RAG proteins in a postcleavage synaptic complex, whose existence helps to explain the known role of RAG1 and RAG2 in the subsequent end-joining events that complete V(D)J recombination.

Base Composition↗
PubMed1997

The interaction of the F plasmid killer protein, CcdB, with DNA gyrase: induction of DNA cleavage and blocking of transcription.

We have studied the interaction of the F plasmid killer protein CcdB with its intracellular target DNA gyrase. We confirm that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (> approximately 160 bp), whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. We show that CcdB, like quinolones, can form a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. We conclude that CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.

Bacterial Proteins↗