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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Duplex-duplex interactions catalyzed by RecA protein allow strand exchanges to pass double-strand breaks in DNA.

Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E. coli RecA protein promotes strand exchanges past double-strand breaks. The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro.

Role of RecA protein spiral filaments in genetic recombination.

Physical and enzymatic studies on RecA protein from Escherichia coli provide the basis for a molecular model of general genetic recombination, a novel feature of which is the role attributed to spiral filaments of RecA protein.

Purification and properties of the recA protein of Proteus mirabilis. Comparison with Escherichia coli recA protein; specificity of interaction with single strand binding protein.

The product of the cloned recA+ gene of Proteus mirabilis substitutes for a defective recA protein in Escherichia coli recA- mutants and restores recombination, repair, and prophage induction functions to near normal levels (Eitner, G., Adler, B., Lanzov, V.

Enzymatic formation of biparental figure-eight molecules from plasmid DNA and their resolution in E. coli.

A key intermediate in general genetic recombination is a structure in which two double-stranded DNA molecules are covalently linked by a single-strand crossover characteristic of a Holliday junction. When the DNA molecules are circular, the recombinant structures take the form of a figure eight. We have used purified E.

Can recA protein promote homologous pairing between duplex regions of DNA?

RecA protein has been shown to promote the formation of joint molecules between intact duplex DNA and homologous gapped DNA. When examined by electron microscopy, such joint molecules display a junction that is, in most cases, distant from the site of the gap. This led us to test whether the observed location of the joint was due to pairing at the gap followed by branch migration, or whether recA-promoted pairing could also take place between duplex homologous regions away from the gap.

Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein.

We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3'-terminus complementary to the single stranded DNA in the gap.

Role of SSB protein in RecA promoted branch migration reactions.

The RecA protein of Escherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of phi X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981 a0. In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle.

Effects of recB21, recF143, and uvrD152 on recombination in lambda bacteriophage-prophage and Hfr by F- crosses.

The effects of the mutation pairs recB21 recF143 and recB21 uvrD152 on the frequency of genetic recombination were investigated in lambda phage-prophage crosses under homoimmune conditions. To prevent recombinants from being formed by the phage red system, these experiments were performed with phages and prophages carrying red and gam mutations. Both spontaneous and damage-induced recombination was measured, the phages being either undamaged or treated with trimethylpsoralen and 360-nm light to cross-link the phage DNA.