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Biomedical subjects

A A Davies

Publications and source records attributed to A A Davies.

At least 19 recordsLinked to original sources
PubMed2001

Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells.

During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or branch migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, branch migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent branch migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted branch migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.

Animals↗
PubMed2001

Role of BRCA2 in control of the RAD51 recombination and DNA repair protein.

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.

Active Transport, Cell Nucleus↗
PubMed1999

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Adenosine Triphosphatases↗
PubMed1999

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.

Adenosine Triphosphatases↗
PubMed1998

Formation of RuvABC-Holliday junction complexes in vitro.

In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair. RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process. Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC). Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected. In the absence of RuvB, RuvAC-junction complexes were observed. Complex formation was not facilitated by duplex DNA. The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction.

Bacterial Proteins↗
PubMed1997

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Adenosine Triphosphatases↗
PubMed1995

Role of the Rad1 and Rad10 proteins in nucleotide excision repair and recombination.

In Saccharomyces cerevisiae, the RAD1 and RAD10 genes are involved in DNA nucleotide excision repair (NER) and in a pathway of mitotic recombination that occurs between direct repeat DNA sequences. In this paper, we show that purified Rad1 and Rad10 interact with a synthetic bubble structure and incise the DNA at the 5'-side of the centrally unpaired region. When Rad1-Rad10 and purified XPG protein (the human homolog of yeast Rad2 protein) were co-incubated with the DNA substrate, we observed incisions at both ends of the bubble. This reaction mimics the dual incision step in nucleotide excision repair in vivo. In addition, the recent suggestion that Rad1 can act to resolve Holliday junctions (Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1994) Nature 371, 531-534), explaining the recombination defect observed in rad1 mutants, has been further investigated. However, using proteins purified in two different laboratories we were unable to show any interaction between Rad1 and synthetic Holliday junctions. The role that Rad1-Rad10 plays in recombination is likely to resemble its activity in NER by acting upon partially unpaired DNA intermediates such as those formed by recombination mechanisms involving single-strand DNA annealing.

DNA Repair↗
PubMed1994

XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair.

Humans with a defect in the XPG protein suffer from xeroderma pigmentosum (XP) resulting from an inability to perform DNA nucleotide excision repair properly. Here we show that XPG makes a structure-specific endonucleolytic incision in a synthetic DNA substrate containing a duplex region and single-stranded arms. One strand of the duplex is cleaved at the border with single-stranded DNA. A cut with the same polarity is also made in a bubble structure, at the 3' side of the centrally unpaired region. Normal cell extracts introduce a nick 3' to a platinum-DNA lesion, but an XP-G cell extract is defective in making this incision. These data show that XPG has a direct role in making one of the incisions required to excise a damaged oligonucleotide, by cleaving 3' to DNA damage during nucleotide excision repair.

Base Sequence↗
PubMed1994

Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase.

A mammalian endonuclease that resolves Holliday junctions has been partially purified from extracts of calf thymus and Chinese hamster ovary cells. The activity acts upon (i) synthetic Holliday junctions and (ii) recombination intermediates made by the Escherichia coli RecA protein and appears to be functionally analogous to the E. coli RuvC protein. Cleavage occurs by the introduction of symmetrically related nicks in strands of like polarity to produce nicked duplex DNA products. The nicks can be repaired by DNA ligase. The resolvase is specific for Holliday junctions and does not act upon Y junctions, G/A mismatches, or heterologous loops. The substrate specificity is therefore similar to that of E. coli RuvC protein and contrasts with the broad range specificity of other junction resolvases such as T4 endonuclease VII. The mammalian resolvase activity has been observed at normal levels in extracts prepared from a series of DNA repair-defective cells. These include the x-ray or UV-sensitive hamster lines xrs-5, xrs-6, and Chinese hamster ovary 43-3B (defective in ERCC-1), and murine cells that are severely immunodeficient and defective in both V(D)J rejoining and DNA repair.

Animals↗
PubMed1994

Tyrosine phosphorylation of alpha tubulin in human T lymphocytes.

N-terminal sequencing of the 55- and 50-kDa polypeptides affinity purified on a phosphotyrosine monoclonal antibody column from activated Jurkat T cells identified alpha and beta tubulin. Two-dimensional gel analysis indicated that alpha tubulin was directly phosphorylated on tyrosine. beta Tubulin was not detectably tyrosine phosphorylated but was precipitated by anti-phosphotyrosine (PTyr) antibody by virtue of its association with the alpha subunit as a heterodimer. Phosphotyrosyl alpha tubulin was not incorporated into intact microtubules and was all in the unpolymerized soluble fraction. These results suggest that tyrosine phosphorylation of alpha tubulin may inhibit the ability of this subunit to polymerize into microtubules. Stimulation of Jurkat T cells via T cell receptor increased the amount of tubulin precipitated by the anti-PTyr antibody. These data raise the possibility that the polymerization of tubulin heterodimers may be regulated by phosphorylation on tyrosine during T cell activation.

Amino Acid Sequence↗
PubMed1992

A growth-dependent post-translational modification of annexin VI.

Annexin VI (p68, 67-kDa calelectrin) is a member of a family of Ca2+/phospholipid-binding proteins, that includes p35 (annexin I) and p36 (annexin II), the major cellular substrates for phosphorylation by the epidermal growth factor receptor and pp60v-src tyrosine kinase activities, respectively. We report here that like annexins I and II, annexin VI is phosphorylated in vivo, but that in contrast, annexin VI phosphorylation is associated with cell growth. In both Swiss 3T3 fibroblasts and human T-lymphoblasts the pattern of phosphorylation followed an almost identical profile. In particular, annexin VI was not phosphorylated in quiescent cells, but was phosphorylated on serine and to a lesser extent threonine, several hours following cell stimulation. Furthermore, annexin VI also incorporated phosphate in a growth-dependent manner, in a form other than a phosphoamino-acid. The phosphate was visualised following acid hydrolysis of immunoprecipitated annexin VI, as part of a complex having high mobility on 2-D thin-layer electrophoresis. The identity of this complex is not known. The results suggest that a post-translational modification other than direct protein phosphorylation may influence the activity of annexin VI and provide evidence linking cell growth with regulation of annexin VI function.

3T3 Cells↗
PubMed1992

CD5 is phosphorylated on tyrosine after stimulation of the T-cell antigen receptor complex.

When T cells are activated by the T-cell antigen receptor, a number of cellular proteins are phosphorylated on tyrosine. We investigated whether any of these proteins were present on the surface of activated T cells. Using the human leukemic T-cell line Jurkat and normal peripheral blood lymphocytes, we identified a 67-kDa cell surface glycoprotein in anti-phosphotyrosine immunoprecipitates, after treatment of the cells with CD3 antibody. When cell lysates were depleted of CD5 by sequential immunoprecipitation, the 67-kDa phosphotyrosyl polypeptide was no longer precipitated by the phosphotyrosine antibody. Western blot analysis of anti-phosphotyrosine precipitates confirmed that this glycoprotein was CD5. It was possible that CD5 was present in the anti-phosphotyrosine immunoprecipitates due to its physical association with phosphotyrosyl proteins rather than being directly tyrosine-phosphorylated itself. However, Western blot analysis of anti-CD5 immunoprecipitates with phosphotyrosine antibody and phosphoamino acid analysis demonstrated that CD5 was indeed phosphorylated on tyrosine after stimulation of the cells with CD3 antibody and was concomitantly phosphorylated on serine and threonine. Tyrosine phosphorylation of CD5 was maximal 2 min after CD3 stimulation and returned to baseline levels by 60 min. CD5 is expressed on the cell surface of all mature T cells and a small proportion of B lymphocytes and has recently been identified as the ligand for CD72, a receptor present on the surface of all B cells. The present data suggest that tyrosine phosphorylation may be involved in B-cell-T-cell communication.

Antigens, CD↗
PubMed1991

The T cell receptor/CD3 complex and CD2 stimulate the tyrosine phosphorylation of indistinguishable patterns of polypeptides in the human T leukemic cell line Jurkat.

Stimulation of the T cell receptor (TcR)/CD3 complex of Jurkat T cells with a monoclonal antibody to the CD3 epsilon chain induced the tyrosine phosphorylation of multiple polypeptides, ranging in size from 21 to 155 kDa. The protein tyrosine phosphorylation was characterized by its rapidity and its transient nature, returning to baseline levels by 60 min. Protein tyrosine kinase activity was also induced when the Jurkat T cells were stimulated with a mitogenic pair of antibodies directed against CD2. Comparison of the polypeptides which were phosphorylated on tyrosine in response to stimulation of the two receptors, by either one- or two-dimensional analysis, failed to reveal any differences. These data suggest that the TcR/CD3 complex and CD2 activated the same tyrosine kinase or kinases. A model is proposed in which CD2 functions as a signal amplifier in physiological responses to antigen/major histocompatibility complex without changing the qualitative nature of the signal generated via the TcR/CD3 complex.

Antibodies, Monoclonal↗
PubMed1989

Evidence that a kinase distinct from protein kinase C induces CD3 gamma-subunit phosphorylation without a concomitant down-regulation in CD3 antigen expression.

An immediate consequence of Ag-specific activation of T cells is phosphorylation of the gamma-subunit of the CD3 gamma-chain. There is good evidence that the kinase that mediates CD3 gamma-chain phosphorylation is protein kinase C (pkC). It has also been proposed that the interaction between pkC and CD3 gamma-chains controls the cell surface expression of the antigen receptor/CD3 Ag complex. In the present study we present data relevant to these two points. Thus we show that CD3 gamma-subunit phosphorylation can be triggered by the calcium ionophore ionomycin. However, as judged by several criteria, ionomycin does not stimulate cellular pkC. Accordingly, ionomycin must regulate phosphorylation of the CD3 Ag by a kinase distinct from pkC. The phosphorylation of CD3 Ag induced by ionomycin is not accompanied by a modulation of the cell surface expression of CD3 molecules which implies that CD3 gamma-chain phosphorylation is not a sufficient signal for the endocytosis of the CD3/Ag receptor complex.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

The gene coding for the p68 calcium-binding protein is localised to bands q32-q34 of human chromosome 5, and to mouse chromosome 11.

The gene coding for human p68, a membrane-associated calcium-binding protein, has been assigned to chromosome 5, using a cDNA clone to probe genomic DNA from rodent-human somatic cell hybrids by Southern hybridisation. The gene was localised, by in situ hybridisation, to 5q32-34. The murine gene was assigned to chromosome 11, using a murine cDNA clone to probe genomic DNA from rodent-rodent somatic cell hybrids.

Animals↗
PubMed1988

Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes.

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide. gamma polypeptide phosphorylated in vitro in GTP gamma S stimulated microsomes incorporated phosphate on Serines 123 and 126. These data are consistent with the involvement of a G protein in the signalling mechanisms that regulate the phosphorylation of the CD3 gamma chain.

Amino Acid Sequence↗
PubMed1987

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.

Amino Acid Sequence↗