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A T Akhmedov

Publications and source records attributed to A T Akhmedov.

At least 19 recordsLinked to original sources
PubMed2000

Human 100-kDa homologous DNA-pairing protein is the splicing factor PSF and promotes DNA strand invasion.

Proteins promoting homologous pairing could be involved in various fundamental biological processes. Previously we detected two mammalian nuclear proteins of 100 and 75 kDa able to promote homologous DNA pairing. Here we report isolation and characterisation of the human (h) 100-kDa DNA-pairing protein, hPOMp100, from HeLa nuclei. The peptide sequences of hPOMp100 revealed identity to the human splicing factor PSF and a DNA-binding subunit of p100/p52 heterodimer of unknown function. Bacterially expressed PSF promotes DNA pairing identical to that of hPOMp100. hPOMp100/PSF binds not only RNA but also both single-stranded (ss) and double-stranded (ds) DNA and facilitates the renaturation of complementary ssDNAs. More important, the protein promotes the incorporation of a ss oligonucleotide into a homologous superhelical dsDNA, D-loop formation. A D-loop is the first heteroduplex DNA intermediate generated between recombining DNA molecules. Moreover, this reaction could be implicated in re-establishing stalled replication forks. Consistent with this hypothesis, DNA-pairing activity of hPOMp100/PSF is associated with cellular proliferation. Significantly, phosphorylation of hPOMp100/PSF by protein kinase C inhibits its binding to RNA but stimulates its binding to DNA and D-loop formation and may represent a regulatory mechanism to direct this multifunctional protein to DNA metabolic pathways.

Base Pairing↗
PubMed1999

Mammalian SMC3 C-terminal and coiled-coil protein domains specifically bind palindromic DNA, do not block DNA ends, and prevent DNA bending.

The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions.

Animals↗
PubMed1999

Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation.

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.

Base Pairing↗
PubMed1999

Human POMp75 is identified as the pro-oncoprotein TLS/FUS: both POMp75 and POMp100 DNA homologous pairing activities are associated to cell proliferation.

We have previously developed an assay to measure DNA homologous pairing activities in crude extracts: The POM blot. In mammalian nuclear extracts, we detected two major DNA homologous pairing activities: POMp100 and POMp75. Here, we present the purification and identification of POMp75 as the pro-oncoprotein TLS/FUS. Because of the pro-oncogene status of TLS/FUS, we studied in addition, the relationships between cell proliferation and POM activities. We show that transformation of human fibroblasts by SV40 large T antigen results in a strong increase of both POMpl00 and TLS/POMp75 activities. Although detectable levels of both POMp100 and TLS/POMp75 are observed in non-immortalized fibroblasts or lymphocytes, fibroblasts at mid confluence or lymphocytes stimulated by phytohaemaglutinin, show higher levels of POM activities. Moreover, induction of differentiation of mouse F9 line by retinoic acid leads to the inhibition of both POMp100 and TLS/POMp75 activities. Comparison of POM activity of TLS/FUS with the amount of TLS protein detected by Western blot, suggests that the POM activity could be regulated by post-translation modification. Taken together, these results indicate that POMp100 and TLS/POMp75 activities are present in normal cells but are connected to cell proliferation. Possible relationship between cell proliferation, response to DNA damage and DNA homologous pairing activity of the pro-oncoprotein TLS/FUS are discussed.

Amino Acid Sequence↗
PubMed1998

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure.

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Base Composition↗
PubMed1998

A B-cell-specific DNA recombination complex.

We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.

Adenosine Triphosphate↗
PubMed1996

SMC proteins constitute two subunits of the mammalian recombination complex RC-1.

Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.

Adenosine Triphosphatases↗
PubMed1996

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei.

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg2+ -dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg2+ -independent.

Adenosine Triphosphatases↗
PubMed1995

Characterization of two nuclear mammalian homologous DNA-pairing activities that do not require associated exonuclease activity.

We have developed an assay to study homologous DNA-pairing activities in mammalian nuclear extracts. This assay is derived from the POM blot assay, described earlier, which was specific for RecA activity in bacterial crude extracts. In the present work, proteins from mammalian nuclear extracts were resolved by electrophoresis on SDS/polyacrylamide gels and then electrotransferred onto a nitrocellulose membrane coated with circular single-stranded DNA (ssDNA). The blot obtained was incubated with a labeled homologous double-stranded DNA (dsDNA). Homologous pairing between the ssDNA and the labeled dsDNA was detected by autoradiography as a radioactive spot on the membrane. In nuclear extracts from mammalian cells, we found two major polypeptides of 100 and 75 kDa, able to promote the formation of stable plectonemic joints. Joint molecule formation required at least one homologous end on the dsDNA, but either end of the dsDNA could be recruited to initiate the reaction. For each polypeptide, the reaction required divalent cations such as Mg2+, Ca2+, or Mn2+. Although ATP was not necessary, ADP was inhibitory in each case. Unlike most of the known eukaryotic DNA-pairing proteins, both activities identified here were able to promote the formation of joint molecules without requiring an associated exonuclease activity. In addition, these two proteins were detected in cell lines from different tissues and from different mammalian species (human, mouse, and hamster).

Adenosine Triphosphate↗
PubMed1995

Characterization of Reca Mediated homologous pairing on nitrocellulose membrane.

Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficient model to study RecA promoted homologous recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this membrane (loading step) which is then incubated with a labelled homologous dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from other organisms, we have characterized the reaction promoted on the membrane. Under these new conditions, the reaction keeps the main characteristics observed with classical assays performed in solution: increasing NaCl concentration destabilized the RecA-DNA complex, ATP gamma S was required for formation of stable RecA-DNA complex, initiation of the reaction exhibits the same polarity as in classical assays, a complete strand exchange with a 44 bp long duplex oligonucleotide has been recorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions involving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATP gamma S only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA (44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiation of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.

Adenosine Triphosphate↗
PubMed1989

[Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts].

The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied. The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells. The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock. The heat-shock induces modification of 88 and 86 Kd heat-shock proteins. It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

[Mutants of Escherichia coli K-12 with increased resistance to ionizing radiation. VI. Increased radioresistance and heat shock proteins].

By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gamr444 and Gamr445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 degrees C and are induced more intensively during heat shock (in comparison to the parental wild-type strain AB1157). When the missense htpR15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into the genome of the Gamr444 mutant, its enhanced radiation-resistance disappeared but could be restored upon introduction of pKV3 plasmid bearing the htpR+ gene. These data show that heat shock proteins are participating in the enhanced radioresistance of Gamr mutants.

DNA Repair↗
PubMed1985

[Hybrid plasmid with bacterial and fungal markers carrying the denV gene of T4 phage and restoring the UV-resistance of E. coli uvrA].

The hybrid plasmid pYBP2 with bacterial (ampR), yeast (LEU2) and bacteriophage T4 (denV) genes has been constructed. The plasmid transformed Escherichia coli CSR603 uvrA recA ampS leuA phr- to ampicillin resistance, leucine independence, UV-resistance similar to the one of uvrA+ recA strain. Cell-free extracts of transformed Escherichia coli cells contain low level of ultraviolet-endonuclease activity in contrast to nontransformed cells containing no enzyme.

Endodeoxyribonucleases↗
PubMed1984

Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae.

Cell-free extracts from wild-type yeast (RAD+) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4 -1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD37 (about 10(4) PD per haploid genome).

DNA, Fungal↗
PubMed1982

Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae.

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca2+ or Mg2+ and AP-endonuclease B by Mg2+ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonucleases B at pH 8. AP-endonuclease A is more stable at 60 degree C (half-life of 17 min) than B (half-life of 4 min). AP-endonuclease A is insensitive to N-ethylmaleimide or rho-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but rho-chloromercuribenzoate inhibits its activity.

Adenosine Triphosphate↗
PubMed1981

Introduction of active enzymes into intact Escherichia coli cells by means of liposomes. Phenotypic suppression of uvr A and pol A mutants.

Genetically deficient cells were supplied with the missing enzymes, purified from an independent source. The introduction of exogenous enzymes into the cells was effected by two independent methods: plasmolysis and liposome transformation. The latter procedure yielded a homogenous cell population which had been rescued from the defect even if the molecular weight of the enzyme amounted to 70 KD (Kilodaltons).

Cell Membrane Permeability↗