Era Cassuto

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

SummaryWe 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. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.

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

SummaryAn 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.

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. To test the latter possibility, intact duplex DNA was incubated with DNA which contained a gap in a region of non‐homology. Joint molecules were detected by filter binding assay and by electron microscopy at about one‐third of the yield observed for fully homologous molecules. These results indicate that initial homologous pairing promoted by recA protein is not restricted to the single‐stranded region in the gap but can also take place in regions where both molecules are duplex.

Regulatory and Enzymatic Functions of recA Protein in Recombination and Postreplication Repair

Investigations into the genetics of recombination, repair and prophage induction in E. coli, have uncovered a remarkable regulatory system involving the lexA gene product as repressor and the recA gene product. This system normally produces lexA and recA proteins at low constitutive levels. The recA protein is a highly specific, single stranded DNA dependent protease capable of cleaving lexA protein and also phage lambda repressor. Postreplication gaps or other damage that produced single stranded regions in the bacterial DNA, may cause recA protein to bind to the DNA. The resulting increase in protease activity leads to the lexA protein being cleaved and thus turns on the transcription of the genes including recA that are under negative control by lexA.

RECENT DEVELOPMENTS IN THE BIOCHEMISTRY OF GENETIC RECOMBINATION

ABSTRACT An in vitro system is described in which the cutting of crosslinked oX RF I DNA molecules by the uvr system of E. coli induces the cutting of homologous undamaged DNA during incubation with crude extracts of thermally induced E. coli (λ precA + ) lysogens. This reaction, which has also been observed in intact E. coli lysogens infected with λ. phages, is dependent on the presence of functional recA + and uvrB + gene products.

Enzymology of Genetic Recombination

Studies of genetic recombination in prokaryotes have shown (1) that recombination occurs by breakage and reunion of DNA, sometimes, but not always, associated with new DNA synthesis, and (2) that the parental contributions to a recombinant molecule are commonly joined by a short heteroduplex or hybrid region. In the past few years, some of the enzymes involved in recombination in prokaryotes have been identified, such as the exonuclease made by bacteriophage λ. Recent studies of λ exonuclease make it possible to rationalize most of the properties of the enzyme in terms of its role in producing a perfect heteroduplex joint between homologous molecules of DNA. λ exonuclease cleaves 5′ mononucleotides from the 5′ end of native DNA in a processive fashion, extensively degrading any molecule of DNA before detaching and attacking another molecule of substrate. The latter property suggests that some control prevents the enzyme from playing an exclusively degradative role. 5′ phosphoryl termini located at gaps in one strand of duplex DNA are resistant to the enzyme. Although 5′ phosphoryl termini at nicks are even more resistant, the enzyme appears to bind weakly at such sites. The significance of these properties may be seen in the enzymes action at the site of a redundant single stranded branch, such as one might expect to find at a joint between two fragments of DNA. A redundant strand is assimilated into the helix, behind λ exonuclease, as the enzyme processively degrades the homologous helical strand. The enzyme recognizes the presence of the redundant strand both for initiation and termination of hydrolysis. Removal of the redundant single strand by the prior action of exonuclease I blocks the action of λ exonuclease on the helical strand. Moreover, when a redundant strand has been completely assimilated through the action of λ exonuclease, the enzyme stops at the precise point which permits the interrupted polynucleotide strand to be sealed by polynucleotide ligase. The sequential action of λ exonuclease and polynucleotide ligase on redundant joint molecules of λ DNA produces intact polynucleotide strands that are biologically active. Several models have been suggested to relate the assimilation of single strands to the genetic recombination of λ and possibly to recombination in other systems as well. Molecules of DNA with double-stranded branches have also been synthesized to test one of the models. The models suggest that λ exonuclease may catalyze a concerted reaction that (1) exposes complementary nucleotide sequences, (2) forms or extends the heteroduplex region, and (3) eliminates redundant branches, precisely restoring a duplex structure that can be sealed covalently by polynucleotide ligase. The λ enzyme, and similar exonucleases, might drive otherwise reversible interactions of a single strand with a recipient duplex, including certain kinds of interactions between two molecules of double-stranded DNA.