Search

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.

The synthesis of an azidoDNA duplex and its use to photolabel DNA polymerases have been previously described (Gibson & Benkovic, 1987). We now present detailed experiments utilizing this azidoDNA photoprobe as a substrate for Escherichia coli DNA polymerase I (Klenow fragment) and the photoaffinity labeling of the protein. The azidoDNA duplex is an efficient substrate for both the polymerase and 3'----5' exonuclease activities of the enzyme. However, the hydrolytic degradation of the azido-bearing base is dramatically impaired. On the basis of the ability of these duplexes to photolabel the enzyme, we have determined that the protein contacts between five and seven bases of duplex DNA. Incubation of azidoDNA with the Klenow fragment in the presence of magnesium results in the in situ formation of a template-primer with the azido-bearing base bound at the polymerase catalytic site of the enzyme. Photolysis of this complex followed by proteolytic digestion and isolation of DNA-labeled peptides results in the identification of a single residue modified by the photoreactive DNA substrate. We identify Tyr766 as the modified amino acid and thus localize the catalytic site for polymerization in the protein. A mansyl-labeled DNA duplex has been prepared as a fluorescent probe of protein structure. This has been utilized to determine the location of the primer terminus when bound to the Klenow fragment. When the duplex contains five unpaired bases in the primer strand of the duplex, the primer terminus resides predominantly at the exonuclease catalytic site of the enzyme. Removal of the mismatched bases by the exonuclease activity of the enzyme yields a binary complex with the primer terminus now bound predominantly at the polymerase active site. Data are presented which suggest that the rate-limiting step in the exonuclease activity of the enzyme is translocation of the primer terminus from polymerase to exonuclease catalytic sites.

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.

Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.

Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used to modify cysteine 907, 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The labeling of cysteine 907 with NBD caused no decrease in the enzyme's polymerase activity, suggesting that the probe did not significantly alter the conformation of the enzyme. The efficiency of singlet-singlet resonance energy transfer was determined from the quantum yield of the donor in the presence and absence of acceptor. By Förster's theory, the measured distances between cysteine 907 and binding sites for duplex DNA, dNTP, and dNMP were 25-39, 19-28, and 17-26 A, respectively. As the fluorophores, attached to the substrates via a tether arm, are separated from the substrates by approximately 12 A, the distances measured between binding sites are subject to this uncertainty. To measure the separation between binding sites for duplex DNA and dNMP, and to reduce the uncertainty introduced by the tether arm, two experiments were carried out. In the first, duplex DNA was labeled with the acceptor fluorophore NBD and used with the donor ANS-modified dNMP to yield a measured distance separating these two sites of 19-28 A.(ABSTRACT TRUNCATED AT 250 WORDS)