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GdnHCl-induced unfolding and reversible folding of beta-lactamase from E. coli have been investigated by measuring enzymatic activity, fluorescence emission and far-UV circular dichroism as indices of the extent of denaturation. The non-coincidence of far-UV CD and fluorescence data and existence of an inflection point clearly suggest the presence of an equilibrium intermediate. The existence of the equilibrium intermediate at around 1 M is corroborated by its enhanced binding of fluorophobic probe 1,8-ANS. The intermediate was found to have a compact shape as measured by its Stokes radius by size-exclusion chromatography. Furthermore, near-UV CD analysis of this enzymatically inactive intermediate showed a significantly disrupted tertiary structure with only a minor change in the secondary structure, which is a characteristic of typical molten globule states. Estimation of the activation energy from the kinetics of unfolding of the protein monitored by fluorescence and CD suggests that the intermediate may be separated from the native and the unfolded state by a high activation-energy barrier.

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g. Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HC1 prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver. The protein-folding activity of E. coli ribosomes was found to be present in 50S particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.

E. coli RecA protein and topoisomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg2+, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When superhelical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages phi X174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of chimeric phage, M13G ori 1, to form catenanes, at least half of which survive heating at 80 degrees C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.

A DNA fragment of 463 base pairs has been isolated by HpaII digestion of the EcoRI cleaved lambdabiot124-10 DNA fragment containing the Escherichia coli biotin gene cluster. This HpaII subfragment contains the biotin regulator region. The biotin regulator DNA has been characterized by electron microscopic heteroduplex analysis, RNA polymerase binding capacity, and the ability to initiate leftward and rightward transcriptions. The two RNA polymerase binding sites are located 63 base pairs and 117 base pairs away from the left end of the HpaII fragment. Both the left- and rightward RNA transcripts have adenosine triphosphates at their 5' end.

Purified recA protein, which is essential for genetic recombination of Escherichia coli, catalyzed ATP-dependent homologous pairing of double-stranded DNA and single-stranded fragments to form D-loops. When the double-stranded DNA was nicked circular DNA (form II) or linear DNA (form III), the reaction proceeded nearly linearly during 30 min of incubation at 37 degrees C. When the double-stranded DNA was superhelical (form I), anomalous kinetics was observed. This anomaly was suppressed by the addition of spermidine without affecting the final yield of D-loops. The formation of D-loops required stoichiometric amounts of recA protein, which were proportional to the concentration of single-stranded DNA but which were not affected by the concentration of double-stranded DNA. With form II or III DNA as the recipient for the formation of D-loops, the rate of the reaction was greatest when there was one monomer of recA protein/2-3 nucleotide residues of single-stranded DNA; larger amounts of single-stranded DNA inhibited the reaction. The formation of D-loops was half inhibited by 30 mM NaCl and by 0.6 mM ADP, one of the products of the reaction. The thermal stability of D-loops made by recA protein was the same as that of D-loops made by annealing. In addition to pairing linear single strands with duplex DNA, recA protein made joint molecules from single-stranded circular DNA and homologous form II or III DNA. According to these and previous observations (Cunningham, R. P., DasGupta, C., Shibata, T., and Radding, C. M. (1980) Cell 20, 223-235), rcA protein will stably pair two molecules of DNA if one of them is single-stranded or partially single-stranded and if either molecule has a free end.

RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends. Stable joint molecules form only when the end of at least one strand is in a homologous region. When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands. The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule. These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions.

Escherichia coli single-strand binding protein (SSB) or phage T4 gene 32 protein reduced the amount of recA protein required to catalyze the formation of D loops from double-stranded DNA and homologous single-stranded fragments. Neither SSB nor gene 32 protein alone catalyzed the formation of D loops, and excessive amounts of either protein, amounts that were sufficient to saturate the single strands, inhibited the formation of D loops completely. Both the stimulatory activity and the inhibitory activity of SSB resisted boiling, which is consistent with the known thermal stability of SSB, whereas the gene 32 protein was inactivated by heating. The formation of D loops in the presence of both recA protein and SSB required homologous DNA and ATP. Spermidine aided the combined action of SSB and recA protein in forming D loops, but Mg2+ alone was sufficient as a counterion.

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.

recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.