Presence Of DNA-binding Protein Research Articles

Thermodynamics and Kinetics of DNA-Protein Interactions from Single Molecule Force Spectroscopy Measurements

When single DNA molecules are stretched, the measurement of the resulting force as a function of extension has yielded new information about the physical, chemical, and biological properties of these important molecules. It has been shown that double-stranded DNA molecules undergo a force-induced melting transition at high forces. Forceextension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review our studies of DNA forceinduced melting in the presence of the classical single-stranded DNA binding protein, gene 32 protein. Bacteriophage T4 gene 32 protein (gp32) is a well studied representative of a large class of single-stranded DNA binding proteins, which are essential for the replication, recombination and repair of DNA. We have developed several new single molecule methods, which when applied to gp32, have led to significant new insights about this proteins structure-function relationships. We discuss a technique for measuring Kss, the association constant of these proteins to ssDNA, which we can determine over a large range of salt concentrations not available to bulk binding studies. In addition, we have measured the noncooperative association constants (Kds) of the weak but biologically-significant interaction with double-stranded DNA as a function of salt concentration for full-length protein and *I, a truncation of gp32 lacking the 48-residue C-terminal domain. Our results have led to a quantitative model for the salt dependence of protein binding, which we postulate to be regulated by a salt-dependent conformational change within the protein involving the C-terminal domain. With this new force spectroscopy technique, we have obtained binding rates and binding free energies for these interactions under a broad range of conditions. Our methodologies should have useful applications in many areas of DNA-protein interactions.

Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase

The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function. The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase. To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity. PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion. Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates. The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed. Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro.

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.

Unwinding and rewinding of DNA by the RecBC enzyme

Under physiological conditions the initial action of the RecBC enzyme (exonuclease V) on duplex DNA is unwinding of the DNA strands. We have examined by electron microscopy the initial products of this unwinding reaction. When such reactions are carried out in the presence of DNA binding protein, unwinding structures are seen both at the terminus of the duplex DNA and at locations remote from the ends of the DNA molecule. Both terminal and internal unwinding structures proceed along DNA at about 300 nucleotides per second, and the single-stranded loops in both types of structure enlarge at about 100 nucleotides per second. In the internal unwindings DNA must be rewound behind the enzyme at about 200 nucleotides per second. The structures do not occur on supercoiled or nicked circular DNA, indicating that free ends are needed for their formation. In the absence of DNA binding protein only internal unwinding structures are seen, suggesting that the internal structures are formed from the terminal unwindings by base-pairing of their unwound single-strand tails. We present a model which incorporates these structures and is consistent with previous observations on the unwinding and degradative actions of the enzyme. In this model the enzyme travels through duplex DNA by unwinding the DNA ahead of itself and rewinding it behind itself. The internal unwindings produced by the RecBC enzyme could be active in the initial synapsis step in genetic recombination.

Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+.

The various activities of the recBC enzyme of Escherichia coli are differentially sensitive to reaction conditions. Although spermidine up to 50 mM and putrescine up to 40 mM in the presence of Mg/sup 2 +/ produce no significant alteration in the activities of the enzyme, low concentrations of Ca/sup 2 +/ have a dramatic effect. In the presence of 1 mM Ca/sup 2 +/, 1 mM Mg/sup 2 +/, and 5 mM ATP, the DNA-dependent ATPase is unaffected, but the double strand DNA exonuclease and the single strand DNA exonuclease and endonuclease activities are completely inhibited. Duplex phage T7 DNA which has been exposed to enzyme under these conditions remains wholly duplex and full length, although about 5 nicks are made randomly in each duplex. When DNA binding protein from E. coli is included in the reaction with enzyme and DNA, the products are, at early stages of the reaction, duplex DNA molecules with one or two single-stranded tails at a terminus; after prolonged incubation, solely single-stranded fragments of 5000 to 35,000 nucleotides are found. (Full length strands are roughly 40,000 nucleotides long.) These products can be rationalized by a model in which the enzyme binds initially to the terminusmore » of a duplex DNA molecule and then tracks along the DNA, unwinding the strands of the helix as it moves. This denaturation would be transient and localized around the enzyme molecule so that the DNA would renature after the enzyme has passed through a particular region. However, the renaturation can be prevented by the presence of DNA binding protein during the reaction. During the passage of an enzyme molecule through a DNA molecule, a limited number of nicks are randomly introduced in a manner essentially unaffected by binding protein. This mode of action is distinct from that observed for the degradation of duplex DNA with Mg/sup 2 +/ but not Ca/sup 2 +/ present.« less

Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.