3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

Abstract

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA. (2) have shown that the formation of joint molecules occurs in such a fashion that only the 5' end of the viral (+)-strand is displaced from the duplex DNA (Fig. 1B). Using the same substrates, Cox and Lehman (3) confirmed this polarity by restriction endonuclease analysis of RecApromoted branch migration. West et al. (4) concluded that the polarity was the same using linearized duplex DNA and homologous circular ss DNA that carried a short hybridized fragment. These data indicate that, in reactions involving linear duplex and ss circular DNA substrates, the polarity of RecA-catalyzed strand exchange in vitro is 5'-to-3' relative to the ss DNA.While the pairing of circular ss DNA with linear duplex DNA is a rapid and efficient reaction whose substrates and products are well characterized, it is not clear what relationship exists between these substrates and those found in vivo. It would be of interest to examine the formation of stablejoint molecules in reactions involving a ss DNA substrate possessing a free end because such a substrate is likely to be more representative of recombinogenic DNA existing in the cell than the ss circular DNAs used in previous in vitro studies (2)(3)(4)(5)(6). In this study, the formation ofjoint molecules between linear ss DNA and circular duplex DNA substrates has been analyzed. These studies reveal that homology at the 3' end of the linear ss DNA is essential for stable RecAcatalyzed joint molecule formation. This observation is consistent with some existing biochemical data (4, 7), which indicate an important role for free homologous 3' ends in recombination. However, it apparently contradicts predictions based on the polarity of strand exchange involving circular ss and linear duplex DNA substrates (2-4): if RecAcatalyzed strand exchange proceeds 5'-to-3' relative to the ss DNA, then linear ss DNAs in which homology to the duplex circle is restricted to the 3' end would form joint molecules that dissociate with time, while those in which homology is present at the 5' end would grow and become more stable.Two possible explanations to resolve this apparent paradox and their relevance to our understanding o...

DNA Strand-Transfer Activity in Pea (Pisum sativum L.) Chloroplasts

“…32P-labeled homologous dsDNA was prepared by digesting supercoiled pUC8 with HaeII, followed by treatment with mung bean nuclease. After separation on 1% agarose gels, the 1.87-kb segment was isolated as described by Tautz and Renz (1983) This method was modified from that of Konforti and Davis (1987). Supercoiled pUC8 was isolated by CsCl/ethidium bromide isopyknic centrifugation (Sambrook et al, 1989).…”

“…coli RecA promotes the exchange of DNA strands between a variety of cDNA substrates. Gel electrophoresis has been used extensively to identify the products of this reaction by their mobility (Konforti and Davis, 1987;Griffith and Harris, 1988;McCarthy et al, 1988). However, in crude cellular extracts, other enzymic activities can lead to artifactual products as discussed in detail by Griffith and Harris (1988).…”

“…In the assays with linear 32P-ssDNA and closed circular dsDNA from pUC8, we used the conditions described by Konforti and Davis (1987) with 10 min of incubation without adding E. coli ssDNAbinding protein. A11 subsequent manipulations were as described above.…”

RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination

“…Recombination initiated by the RecBCD enzyme begins from such a blunt dsDNA end present at a dsDNA break, produced as the result of DNA damage, a broken replication fork, or conjugal DNA transfer. After binding to this end, the helicase and nuclease activities of RecBCD enzyme convert the dsDNA to a 3Ј-ssDNA overhang, a product that is the preferred substrate for RecA protein-mediated strand invasion (Konforti and Davis 1987;Anderson and Kowalczykowski 1997a,b;for review, see Kowalczykowski and Eggleston 1994). Because RecQ helicase also acts on blunt dsDNA ends and is needed for recombination in a recBCsbcBC background, it was proposed to act as a functional analog of RecBCD enzyme during this processing step (Clark and Sandler 1994;.…”

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial GeneraCampylobacterandHelicobacter

“…Once a region suitable for recombination is found, RecA initiates a DNA strand exchange reaction that forms a recombination intermediate or joint molecule (Fig. 5) (83,335,345,346,505,638,639). Formation of the joint molecule results in the development of a mobile DNA heteroduplex known as a Holliday junction (146,264,345).…”