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P Howard-Flanders

Publications and source records attributed to P Howard-Flanders.

At least 19 recordsLinked to original sources
PubMed1990

RecA-ssDNA interaction: induced strand cleavage by hydroxyl radical at a defined distance from the 5' end.

Interaction of the RecA protein with single-stranded DNA (ssDNA) was analyzed by challenge with the hydroxyl radical, which can cleave the DNA backbone. We found that RecA protein induces cleavage by the radical at a defined distance from the 5' end. The cleavage was at the 11th nucleotide in many oligodeoxynucleotides. Cleavage may be intermittent since a second cleavage was induced at the 22nd or 21st site. This specific cleavage was observed under optimal conditions for filament formation, homologous pairing and strand exchange. Specificity in cleavage was, however, decreased by replacement of ATP by adenosine 5'-(gamma-thio)triphosphate (ATP gamma S), replacement of RecA protein by a mutant (RecA1) protein, or an increase in Mg2+ concentration. We propose that RecA protein induces a special structural alteration, such as bending, perhaps sequentially, on ssDNA and that this altered site plays an important role in homologous pairing and strand exchange.

Adenosine Triphosphate↗
PubMed1990

DNase I footprinting analysis of RecA protein polymerized on DNA during strand exchange reaction between a gapped circle and a linear duplex.

RecA protein mediates homologous pairing and strand exchange reactions between a circular duplex with a single strand gap and a linear duplex. We have used the DNase I footprinting method to analyze processes involving four strands during these reactions. We asked how the length of DNA protected by RecA protein changes as these reactions proceed. We compared two kinds of gapped DNAs. We found that RecA protein polymerizes rapidly in the forward direction (5' to 3' with respect to the single strand). We found, however, that polymerization in the reverse direction was more prominent with a duplex carrying a longer gap than one carrying a shorter gap. DNase I footprints showing protection by RecA protein were obtained only at limited nuclease concentrations, which in turn depended on the position of the end label and the stage of the strand exchange reaction. As judged by the concentrations of DNase I good for footprinting, the extent of protection by RecA protein was greatest for (+) single-stranded DNA at its first binding site, next highest for heteroduplex containing this (+) strand, and least for the gapped homoduplex DNA. These differences in DNase I sensitivity can be explained in terms of differences in the accessibility of various strands on the basis of a three-dimensional model for the strand exchange reaction.

Binding Sites↗
PubMed1989

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I.

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.

Cross-Linking Reagents↗
PubMed1989

Proteins induced by DNA-damaging agents in cultured Drosophila cells.

In Drosophila cultured cells, the effects of several DNA-damaging agents on the expression of proteins were investigated. Poly(A+) RNA prepared from both untreated cells and cells treated with DNA-damaging agents was translated in vitro. The translation products were analyzed by two-dimensional electrophoresis. Methyl methanesulfonate, the most potent agent used, induced about 25 proteins, some new and some enhanced pre-existing proteins. Angelicin plus near UV irradiation, 4-nitroquinoline N-oxide and ethyl methanesulfonate were efficient inducers. Mitomycin C, UV irradiation and hydrogen peroxide were poor inducers, inducing only a few proteins at low levels. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, and a DNA gyrase inhibitor, nalidixic acid, also were used. In this system they were weak inducers of new proteins. Several of the new or enhanced proteins were common to several agents, but others were agent specific. The distribution of mutagen-induced proteins was compared with that of proteins induced in cells heated at 37 degrees C. Some of the proteins induced by DNA-damaging agents were found to overlap heat-shock proteins. These results suggest that there are sets of induced genes that are regulated differently.

Animals↗
PubMed1989

Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.

4,5',8-Trimethylpsoralen (psoralen) plus near UV light produces interstrand crosslinks and monoadducts in DNA, both of which are mutagenic. In Escherichia coli, crosslinks are incised by UvrABC excinuclease, an event that can lead to homologous recombination and repair. To determine whether UvrABC incision of crosslinks is a step in the path to mutagenesis as well as repair, the effect of DNA homologous to a target gene on a plasmid was determined. pSV2-gpt DNA was treated with psoralen and transformed into a pair of hosts: one was gpt+, the other was delta (gpt-lac)5. The DNA was extracted and transformed into a tester strain [delta (gpt-lac)5] in which Gpt- mutations in the plasmid were scored. The results show that psoralen-induced mutations were reduced to background levels by the presence of the gpt+ homolog in the host chromosome. delta gpt hosts that were constitutively induced for the SOS response yielded point mutations, whereas noninduced hosts yielded almost exclusively large deletions. Since crosslinks were estimated to be responsible for most of the mutations observed, we conclude that the premutagenic lesion of psoralen crosslinks is recombinagenic and therefore very likely to be the product of UvrABC incision.

Cross-Linking Reagents↗
PubMed1988

Structure of helical RecA-DNA complexes. III. The structural polarity of RecA filaments and functional polarity in the RecA-mediated strand exchange reaction.

The RecA protein of Escherichia coli has been used in vitro to mediate a strand-exchange reaction between homologous DNA molecules. A three-dimensional reconstruction of a RecA filament on double-stranded DNA has been previously determined from electron micrographs, and the reconstruction displays a clear axial polarity. The RecA-mediated strand-exchange reaction between a double-stranded DNA and a homologous single-stranded DNA that is complexed with a RecA helical polymer proceeds with a known polarity. Using image analysis of electron micrographs, we have determined the relation between the structural polarity of RecA filaments and the 3' and 5' polarity of single-stranded DNA. Thus, the structural polarity of RecA filaments can now be related to the direction in which the RecA-mediated strand-exchange reaction advances along the complexed single-stranded DNA.

DNA, Bacterial↗
PubMed1988

RecA-mediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions, and insertions.

RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded. Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends. The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules. When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex. In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light. Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA. However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection. These results contrast with those obtained in related three-stranded reactions (Bianchi, M. E., and Radding, C. M. (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.

Base Sequence↗
PubMed1987

Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP.

An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from HeLa cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.

Adenosine Triphosphatases↗
PubMed1986

The binding of RecA protein to duplex DNA molecules is directional and is promoted by a single stranded region.

RecA protein from E. coli binds more strongly to single stranded DNA than to duplex molecules. Using duplex DNA that contains single stranded gaps, we have studied the protection by RecA protein at various concentrations, of restriction sites as a function of their distance from the single stranded region. We show that the binding of RecA protein, initiated in the single stranded region, extends progressively along the adjoining duplex in the 5' to 3' direction with respect to the single stranded region. The strand exchange reaction is known to proceed in the same direction.

DNA, Circular↗
PubMed1985

Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase.

Hexamine cobalt chloride (HCC) increases the efficiency of blunt end ligation by T4 DNA ligase about 50 fold. Maximum stimulation occurs when standard buffers for ligation are supplemented with 1 mM HCC. All the ligation events are intermolecular regardless of the initial DNA concentration. In the presence of monovalent cations (eg. 25 mM KCl) HCC still increases the extent of T4 catalyzed ligation but intramolecular ligation products are also formed. Therefore, intermolecular ligation can be performed rapidly and at low DNA concentrations.

Cations, Monovalent↗
PubMed1985

Isolation of altered recA polypeptides and interaction with ATP and DNA.

In this paper we describe the partial proteolytic digestion of recA proteins from Escherichia coli and Proteus mirabilis and the production and isolation of truncated recA polypeptides. A proteolytic fragment of the P. mirabilis recA protein bound single-strand DNA and ATP normally but has altered duplex DNA binding properties. This protein was shown to initiate but not complete DNA strand transfer from a DNA duplex to a complementary single strand. The product of the E. coli recA1 allele bound but could not hydrolyze ATP and the protein bound single-strand but not double-strand DNA. This protein did not appear to initiate the transfer of a strand from a linear duplex to a single-strand circle and inhibited the wild-type recA protein from performing strand transfer. We report that recA protein binds linear duplex DNA in a manner that enhances the rate of ligation by T4 DNA ligase. When heterologous single-strand DNA was added in addition to the duplex DNA large stable aggregates of protein and DNA were formed that could easily be sedimented from solution.

Adenosine Triphosphate↗
PubMed1979

The isolation and genetic characteristics of lambda transducing phages of the uvrA+ and uvrC+ genes of E. coli K12.

Lambda transducing phages carrying the excision repair genes uvrA+ and uvrC+ were selected from a pool of lambda phages carrying EcoR1 fragments of E. coli DNA. These phages and also lambdauvrB+ (obtained from Gottesman) were used to make lysogens of excision-defective strains carrying uvrA-, uvrB- or uvrC-. Lambda uvrA+ was found to transduce strains carrying uvrA- but not those carrying uvrB- or uvrC-, to normal ultraviolet resistance. Similarly, lambdauvrB+ and lambdauvrC+ were found to complement only the corresponding uvr- allele. The lambda transducing phages were co-transduced with gal+ by P1 phage into lysogenic gal- recipients, and presumably were integrated at the normal prophage site.

Alleles↗
PubMed1978

Homology-dependent cutting in trans of DNA in extracts of Escherichia coli: an approach to the enzymology of genetic recombination.

An in vitro system is described in which the cutting of crosslinked phiX replicative form (RF) I DNA molecules by the uvr system of Escherichia coli induces the cutting of homologous undamaged DNA during incubation with crude extracts of thermally induced E. coli (lambda precA+) lysogens. This reaction, which has also been observed in intact E. coli lysogens infected with lambda phages, is dependent on the presence of functional recA+ and uvrB+ gene products. Extracts from thermally induced lambda precA+ lysogens of E. coli proved to be substantially more active than extracts from nonlysogenic cells of the same strain. The results provide preliminary evidence for an endonuclease activity that cuts intact superhelical DNA in response to interaction with homologus damaged DNA. In the present paper, we describe an in vitro system in which both the endonucleolytic cutting of DNA containing crosslinks and the induced cutting of undamaged DNA can be studied without purification of the participating enzymes. Although the information obtained is fragmentary and often puzzling, we feel that this system can contribute to an understanding of the complex mechanisms involved in repair and recombination.

Coliphages↗
PubMed1977

Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and tri-methylpsoralen or khellin.

The furocoumarin, 4,5',8-trimethylpsoralen, sensitizes cells and viruses to 360 nm light, producing cross-links and monoadducts in their DNA. The furanochromone khellin is a less effective sensitizing agent than psoralen, but has been found to induce cross-links and adducts in DNA also. The number of cross-links increases as the square of the time of exposure to light. We found that greater fluences were required for khellin than for psoralen, possibly because of the less favorable angle of the distal unsaturated bonds for corss-linking pyrimidines in adjacent base pairs. By adjusting the time of exposure to 360 nm light, lambda phages were damaged with [3H]psoralen and [3H]khellin so as to produce equal numbers of cross-links. These exposures were found to produce 8-times more [3H]khellin than [3H]psoralen adducts in the DNA of the phages. Similar exposures were made with nonradioactive photosensitizers to determine the effectiveness of lambda phages carrying cross-links and monoadducts in producing genetic recombinants. Lambda phage-prophage genetic corsses were performed with psoralen and khellin-damaged phages under repressed conditions in which replication of the damaged DNA was blocked. It was estimated from the results that cross-links were about 20-times more effective than monoadducts for inducing recombination under repressed conditions. In tests on the survival of plaque forming ability on wild type bacteria, it was estimated that cross-links were about 15-times more effective than the adducts. The results support the conclusion that, in homoimmune crosses with psoralen-damaged lambda phages infecting wild type lysogens, more than three-quarters of the induced recombination can be attributed to cross-links rather than to monoadducts.

Binding Sites↗