Surendran Rajendran

Does Single-stranded DNA Pass through the Inner Channel of the Protein Hexamer in the Complex with the Escherichia coli DnaB Helicase? FLUORESCENCE ENERGY TRANSFER STUDIES

The structure of the complex of theEscherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5′ arm of the replication fork, without ATP hydrolysis, the distance between the 5′ end of the arm and the 12-kDa domains of the hexamer (R = 47 Å) is the same as in the complex with the isolated ssDNA oligomer (R = 47 Å) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5′ arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2–3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.

Functional and Structural Heterogeneity of the DNA Binding Site of the Escherichia coli Primary Replicative Helicase DnaB Protein

The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques. Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site. The total site is built of two subsites differing in structure and affinity, although both occlude ∼10 nucleotides. ssDNA affinity for the strong subsite is ∼3 orders of magnitude higher than that for the weak subsite. Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone. This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution. The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5′-end of the ssDNA. The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite. The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.

Human DNA Polymerase β Recognizes Single-stranded DNA Using Two Different Binding Modes

Interactions between the human DNA polymerase β (pol β) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(dεA)) as a function of the binding density of pol β-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol β forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol β binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol β)16 and the second as (pol β)5binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol β binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol β, is discussed.

Studies of Macromolecular Interaction by Sedimentation Velocity

As our knowledge expands and new systems are investigated, it becomes clear that elucidating precise biochemical regulatory mechanisms requires detailed understanding of macromolecular assembly processes. For example, the regulation of gene expression involves an intricate network of protein-protein and protein-nucleic acid interactions and the mechanism of some allosteric enzymes is linked to subunit assembly. In order to define these mechanisms one needs information on the identities of proteins in the complex, the affinities of these proteins for each other and the effects of regulators on the formation of these complexes. A direct way of studying macromolecular assembly is to monitor the resulting changes in mass as a consequence of the formation of these macromolecular complexes. One of the methods that enables one to directly monitor the mass of macromolecules is the transport technique. Among the transport methods sedimentation analysis is the technique of choice because of the sound fundamental principles on which the method is based and because of its resolving power. Excellent reviews on the applications of sedimentation equilibrium in studying macromolecular self-associations and heteropolymers formation are included in Part I of this volume. In this chapter the focus is on applications of sedimentation velocity. One of the advantages of sedimentation velocity over that of equilibrium is its speed of analysis, e.g., a run can be completed within an hour whereas equilibrium experiments may take much longer. Hence, if a biological sample is unstable the study on that system may have to be conducted using sedimentation velocity. However, a larger amount of sample will be required for velocity analysis than for equilibrium measurements, e.g., the calf brain tubulin and rabbit muscle phosphofructokinase systems require a few mg of protein to define a curve of sedimentation coefficient vs concentration.

T4 ENDONUCLEASE V EXISTS IN SOLUTION AS A MONOMER AND BINDS TO TARGET SITES AS A MONOMER

Endonuclease V, a N-glycosylase/lyase from T4 bacteriophage that initiates the repair of cyclobutane pyrimidine dimers in DNA, has been reported to form a monomer-dimer equilibrium in solution [Nickell and Lloyd (1991) Biochemistry 30, 8638], although the enzyme has only been crystallized in the absence of substrate as a monomer [Morikawa et al. (1992) Science 256, 523]. In this study, analytical gel filtration and sedimentation equilibrium techniques were used to rigorously characterize the association state of the enzyme in solution. In contrast to the previous report, at 100 mM KCl endonuclease V was found to exist predominantly as a monomer in solution by both of these techniques; no evidence for dimerization was seen. To characterize the oligomeric state of the enzyme at its target sites on DNA, the enzyme was bound to oligonucleotides containing a single site specific pyrimidine dimer or tetrahydrofuran residue. These complexes were analyzed by nondenaturing gel electrophoresis at various acrylamide concentrations in order to determine the molecular weights of the enzyme-DNA complexes. The results from these experiments demonstrate that endonuclease V binds to cyclobutane pyrimidine dimer and tetrahydrofuran site containing DNA as a monomer.