Recognition of duplex DNA containing single-stranded regions by recA protein.

Abstract

Genetic recombination in Escherichia coli requires recA protein, the product of the recA+ gene. In this paper we show that purified recA protein, which binds strongly to denatured DNA, cooperatively recognizes DNA containing short single-stranded regions. The interaction of varying amounts of recA protein with DNA molecules was investigated by measuring its DNA-dependent ATPase activity. In 3 mM Mg2+, the ATPase activity was stimulated by excess singlestranded DNA The recA + gene product plays an indispensable role in homologous genetic recombination in Escherichia coli. This protein has been identified (1-3) and purified to near homogeneity (4, 5). It is produced in large amounts in bacteria after treatments that damage DNA (6, 7). It exists in monomeric and tetrameric forms, dependent upon protein, salt, and ATP concentrations (8); it has a protease activity that cleaves X repressor (9-11) and an ATPase activity that is dependent upon single-stranded DNA and Mg2+ (4,5,8). Two recent studies have shown that recA protein catalyzes the ATP-dependent hybridization of single-stranded DNA to homologous duplex molecules. The product of this reaction is a D loop in which the single strand is hydrogen bonded to one of the duplex strands (5, 12).Because postreplication gaps in the DNA of UV-irradiated bacteria induce sister-strand exchanges with an efficiency close to unity, it appears that the enzymes of genetic recombination may recognize single-stranded gaps as substrate (13). These observations prompted us to investigate the interaction of varying amounts of recA protein with duplex DNA containing short single-stranded gaps. In this paper, we demonstrate that recA protein recognizes gapped duplex DNA and forms complexes that %timulate the ATPase activity. The significance of this interaction with respect to homologous pairing is discussed.MATERIALS AND METHODS Preparation of Gapped DNA. 3H-Labeled X cI DNA was prepared as described (14) and dissociated to the monomeric form by heating to 55°C for 2 min followed by quick cooling prior to use. Single-stranded regions were digested by incubation with nuclease S1 in 2-ml reaction mixtures containing 160 Mug of X DNA, 100 mM NaCl, 50 (18). recA protein was recovered by Polymin P fractionation from 100 g of nalidixic acid-induced cells as described (4). After precipitation with (NH4)2SO4 at 0.25 g/ml, the protein was dialyzed against 20 mM sodium phosphate, pH 6.8/10% (vol/vol) glycerol/10 mM 2-mercaptoethanol and applied to a hydroxylapatite column (Bio-Gel HPT). The column was eluted with a 20-300 mM phosphate gradient and, after dialysis, the recA protein was loaded onto a DEAE-cellulose column equilibrated with 20 mM Tris1HCl, pH 7.5/0.5 mM EDTA/50 mM NaCl/10% glycerol/i mM dithiothreitol. After elution with a salt gradient, recA protein was further purified by gel filtration through Bio-Gel A-0.5m agarose. Storage was at -70°C in 50 mM Tris-HCI, pH 7.5/0.1 mM EDTA/50 mM NaCI/50% glycerol/1 mM dithiothreitol.35S-Labeled recA protein was prepared from b...

Molecular Cloning of a recA-Like Gene from the Cyanobacterium Anabaena Variabilis

“…The invasion of duplex DNA by single-stranded homologous DNA is stimulated in vitro by the addition of purified recA protein (3). Other studies support the conclusion that with the addition of ATP, this protein facilitates reciprocal strand transfer between two double helices (18).…”

Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

“…In vitro studies revealed the following steps for the formation of the joint molecules by RecA protein: (i) in the presence of ATP (or its unhydrolyzable analogue, ATP␥S), RecA protein cooperatively polymerizes along single-stranded DNA (15)(16)(17), resulting in the formation of ''presynaptic filaments'' in which the DNA is extended up to 1.5-fold compared with B-form DNA with the same sequence (18,19); (ii) the presynaptic filament captures double-stranded DNA to search for homology to the singlestranded DNA present in the filament and upon homologous alignment of the DNA molecules a core heteroduplex is formed (the completion of homologous pairing; 15,18,20,21); and (iii) the core heteroduplex is unidirectionally extended and stabilized by strand exchange (12,22,23; see also refs. 24-30 for review).…”

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding

“…la), but not to cover all the double stranded regions for DNA concentrations higher than 5 ug/ml. We have previously suggested that recA protein binds to single stranded DNA and spreads onto the adjoining duplex in a gapped molecule (6).…”

“…The purification of recA protein has been described (6 1.25 ;zg of gapped 3H-DNA were incubated with 0.1 to 0.8 mg/ml of recA protein in 50 pl of 33 mM Tris. HCl, pH 7.5, 25 mM MgCl2, 2mM dithiothreithol, 100 pg/ml bovine serum albumin (BRL) and 1.2 mM ATP.…”

“…This reaction is interesting because it resembles the homologous pairing between two duplexes in genetic recombination or postreplication repair in intact cells. Moreover, since postreplication gaps cause exchanges with an efficiency close to unity (5), it appears that gapped duplexes may be a natural substrate for recA protein (6).…”