Search PubMed⌕ Search

Biomedical subjects

R Fishel

Publications and source records attributed to R Fishel.

At least 73 records · Page 4Linked to original sources
PubMed1994

Binding of mismatched microsatellite DNA sequences by the human MSH2 protein.

Alteration of the human mismatch repair gene hMSH2 has been linked to the microsatellite DNA instability found in hereditary nonpolyposis colon cancer and several sporadic cancers. This microsatellite DNA instability is thought to arise from defective repair of DNA replication errors that create insertion-deletion loop-type (IDL) mismatched nucleotides. Here, it is shown that purified hMSH2 protein efficiently and specifically binds DNA containing IDL mismatches of up to 14 nucleotides. These results support a direct role for hMSH2 in mutation avoidance and microsatellite stability in human cells.

Base Composition↗
PubMed1994

Purified human MSH2 protein binds to DNA containing mismatched nucleotides.

The human hMSH2 protein is a member of a highly conserved family of postreplication mismatch repair components found from bacteria to humans. Alterations of the gene coding for this protein cosegregate with, and are the likely cause of, chromosome 2-linked hereditary nonpolyposis colon cancer. Postreplication mismatch repair has been found to faithfully replace misincorporated nucleotides, thereby increasing the overall fidelity of DNA replication. Loss of postreplication mismatch repair function leads to a mutator phenotype, which is proposed to account for the multiple mutations required for multistep carcinogenesis. Although the functions of hMSH2 can be anticipated based on its similarity to well-characterized bacterial and yeast proteins, proof of its functions has not been established. Here we demonstrate that purified hMSH2 binds specifically to mismatched nucleotides, providing a target for the excision repair processes characteristic of postreplication mismatch repair.

Base Sequence↗
PubMed1994

Nonhomologous recombination in human cells.

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

Adenoviridae↗
PubMed1993

The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer.

We have identified a human homolog of the bacterial MutS and S. cerevisiae MSH proteins, called hMSH2. Expression of hMSH2 in E. coli causes a dominant mutator phenotype, suggesting that hMSH2, like other divergent MutS homologs, interferes with the normal bacterial mismatch repair pathway. hMSH2 maps to human chromosome 2p22-21 near a locus implicated in hereditary nonpolyposis colon cancer (HNPCC). A T to C transition mutation has been detected in the -6 position of a splice acceptor site in sporadic colon tumors and in affected individuals of two small HNPCC kindreds. These data and reports indicating that S. cerevisiae msh2 mutations cause an instability of dinucleotide repeats like those associated with HNPCC suggest that hMSH2 is the HNPCC gene.

Amino Acid Sequence↗
PubMed1991

The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A.

Homologous pairing and strand exchange of DNA are catalyzed by the human homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires homologous DNA substrates and stoichiometric quantities of HPP-1. Here we show that the addition of the purified human single-strand binding (SSB) protein hRP-A to the reaction mixture stimulates the rate of homologous pairing 70-fold and reduces the amount of HPP-1 required for the reaction at least 10-fold. The identification of hRP-A as a stimulatory factor of HPP-1-catalyzed reaction was facilitated by its recognition as a member of a high molecular weight complex of recombination components. Neither the Escherichia coli SSB protein, bacteriophage T4 gene 32 protein, nor the highly conserved Saccharomyces cerevisiae yRP-A SSB protein could substitute for hRP-A in this stimulation. Because only the cognate SSB was capable of stimulating HPP-1, these results suggest that eukaryotes depend on unique and specific interactions between DNA recombination components.

Blotting, Western↗
PubMed1991

Evi-1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein.

Evi-1 was originally identified as a common site of viral integration in murine myeloid tumors. Evi-1 encodes a 120-kDa polypeptide containing 10 zinc finger motifs located in two domains 380 amino acids apart and an acidic domain located carboxy terminal to the second set of zinc fingers. These features suggest that Evi-1 is a site-specific DNA-binding protein involved in the regulation of RNA transcription. We have purified Evi-1 protein from E. coli and have employed a gel shift-polymerase chain reaction method using random oligonucleotides to identify a high-affinity binding site for Evi-1. The consensus sequence for this binding site is TGACAAGATAA. Evi-1 protein specifically protects this motif from DNase I digestion. By searching the nucleotide sequence data bases, we have found this binding site both in sequences 5' to genes in putative or known regulatory regions and within intron sequences.

Animals↗
PubMed1990

Purification and characterization of a protein from human cells which promotes homologous pairing of DNA.

A human protein of approximately 120 kilodaltons has been purified to homogeneity based on its ability to catalyze the homology-dependent transfer of the complementary strand from a linear duplex DNA to a circular single-strand DNA. The activity was purified from an immature T-cell acute leukemic tumor cell line, with the majority of enrichment obtained by chromatography on a novel Z-DNA affinity column. The human homologous pairing protein was found to absolutely require homologous DNA substrates in a reaction that needs nearly stoichiometric amounts of protein. The homologous pairing activity is not stimulated by addition of exogenous ATP; however, the photo-cross-linking ATP analog 8-azidoadenosine 5'-[32P] triphosphate (8-N3-[32P]ATP) binds specifically to the homologous pairing protein. Electron microscopic analysis demonstrated the formation of all expected products. Intermediate strand-exchange products were shown to conserve the displaced DNA strands, eliminating many alternate explanations for the homologous pairing activity. These and other biochemical properties described in this report suggest that the nature of homologous pairing by the human protein is functionally similar to that of the bacterial RecA protein, although the exact mechanism of strand exchange may be somewhat different.

Adenosine Triphosphate↗
PubMed1990

Z-DNA affinity chromatography.

In this chapter we have detailed a method that can be generalized to link virtually any DNA substrate to a chromatography matrix at its ends via an avidin-biotin linkage. We have used this technique to construct a left-handed Z-DNA column for the purpose of identification and purification of Z-DNA-binding proteins. This technique for the linkage of DNA to a column matrix by avidin-biotin technology can be modified, however, to produce linked multimeric sequences specific for regulatory or other DNA-binding proteins.

Avian Myeloblastosis Virus↗
PubMed1990

Circular single-stranded RNA replicon in Saccharomyces cerevisiae.

Circular RNA replicons have been reported in plants and, in one case, in animal cells. We describe such an element in yeast. In certain yeast strains, a 20S RNA species appears on transfer of cells to acetate medium. This phenotype shows cytoplasmic (non-Mendelian) inheritance and the 20S RNA is associated with 23-kDa protein subunits as a 32S particle. We demonstrate that yeast 20S RNA is an independent replicon with no homology to host genomic, mitochondrial, or 2-microns plasmid DNA or to the L-A, L-BC, or M1 double-stranded RNA viruses of yeast. The circularity of the 20S RNA is shown by the apparent absence of 3' and 5' ends, by two-dimensional gel electrophoresis, and by electron microscopy. Replication of yeast 20S RNA proceeds through an RNA-RNA pathway, and a 10,000-fold amplification occurs on shift to acetate medium. The copy number of 20S RNA is also reduced severalfold by the SKI gene products, a host antiviral system that also lowers the copy numbers of yeast double-stranded RNA viruses. Yeast 20S RNA and the hepatitis delta virus show some similarities.

Genotype↗
PubMed1990

recB recC-dependent processing of heteroduplex DNA stimulates recombination of an adjacent gene in Escherichia coli.

The effect of DNA mismatched repair on the genetic recombination of a gene adjacent to the mismatch site (MS) was tested by using four mismatch configurations. An MS was constructed in a well-characterized plasmid recombination substrate, and recombination with a resident compatible plasmid was measured after transformation of the mismatched plasmid into Escherichia coli. The mismatched plasmids were constructed such that one of the DNA strands was methylated by the DNA adenine methylase (Dam), while the other strand was unmethylated. The processing of a hemimethylated single-base-pair mismatch had no effect on the recombination of the adjacent gene, suggesting that the most efficient (Dam-instructed) mismatch repair process does not secondarily promote genetic recombination. However, mismatches that could form an ordered secondary structure resembling a cruciform increased the recombination of this adjacent gene at least 20-fold. An identical mismatch that could not form an ordered secondary structure had no effect in this system. The increased frequency of recombination observed was found to require the recB or recC gene product or both. Furthermore, the recombination appeared unidirectional, in that the cruciform-containing plasmid did not produce stable transformants. Our results support a model in which the cruciform-containing plasmid can participate in recombination with the resident plasmid but is unable to produce stable transformant progeny. A proposed role for the RecBCD enzyme (ExoV) in this process is discussed.

DNA Repair↗
PubMed1989

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.

Bacterial Proteins↗
PubMed1989

The human recombination strand exchange process.

A mechanism for the initiation of general recombination that involves the formation of left-handed Z-DNA heteroduplex segments adjacent to right-handed B-DNA heteroduplex segments is discussed. The paranemic nature of this initiation structure allows for homology recognition in the absence of strand cleavage. This model suggests that proteins catalyzing recombination initiation via the formation of paranemic joint should in some capacity recognize Z-DNA. Other studies have shown that both the RecA protein of Escherichia coli and the Rec1 protein of Ustilago maydis have a greater affinity for Z-DNA than B-DNA. Here we have used Z-DNA affinity chromatography to purify a peptide of approximately 120 kilodaltons from a human tumor cell line that catalyzes a simple recombination strand-transfer reaction similar to one developed for the characterization of the RecA and Rec1 proteins. We report details of the characterization of the human strand-transfer activity and identified a potential human recombination complex.

Affinity Labels↗
PubMed1985

Cocaine colitis. Is this a new syndrome?

An unusual case of colitis in a 37-year-old cocaine addict is described. The patient presented with right-sided abdominal pain and diarrhea exacerbated by his use of cocaine. Significant antibiotic ingestion was denied. At laparotomy, an edematous cecum and ascending colon were found, the cut surface of which revealed diffuse superficial ulcerations and yellowish fibrinous material. Microscopic examination demonstrated findings consistent with pseudomembranous colitis with an ischemic component. A mechanism involving catecholamine-induced mucosal ischemia is postulated to explain the findings seen in this patient.

Adult↗
PubMed1983

Cyclosporine A impairs wound healing in rats.

Cellular immune responses may play an important role in the early inflammatory and cellular phases of wound healing. Cyclosporine A (CSA), a new immunosuppressive agent, impairs cellular immunity and T-cell-dependent humoral immunity. Therefore, the effect of CSA-induced immunosuppression in a rat wound-healing model was studied. Sprague-Dawley rats underwent a standardized skin incision and subcutaneous implantation of sterile polyvinyl alcohol sponges. CSA was dissolved in olive oil and given by gavage to one group of animals at a total dose of 125 mg/kg/10 days. The control group received an equivalent volume of olive oil. Ten-day-old wounds were weaker in CSA-treated animals, both in the fresh state (282 +/- 19 g vs 380 +/- 27 g, P less than 0.01), and after formalin fixation (1111 +/- 74 g vs 1419 +/- 57 g, P less than 0.01). In addition, CSA-treated rats accumulated significantly less hydroxyproline in the wound sponge granuloma, an index of reparative collagen deposition. The impairment in wound healing occurred without differences in body weight gain or organ weights. There was a profound immunosuppression in the animals receiving CSA as determined by thymic lymphocyte blastogenesis in response to Con A and PHA. These findings suggest that immunosuppression in otherwise healthy animals impairs wound healing.

Adrenal Glands↗