Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy

Abstract

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.

Function and Structure Relationships in DNA Polymerases

“…The labeling of Tyr766 by a photoaffinity probe at the primer terminus is likewise consistent with the general location of the polymerase catalytic site (57). Chemical footprinting (58), fluores cence (59,60), and photocrosslinking (57) experiments together indicate that 5-8 basepairs of duplex DNA are covered by Klenow fragment when the primer terminus is at the polymerase site. The range of values probably reflects, on the one hand, the particular requirements for access of the footprinting reagent and, on the other, uncertainties as to the precise orientation of fluorescent or photoactivatable probes attached to DNA; again, the data are consistent with the proposed model but are insufficiently discriminating to rule out alternatives that could be proposed.…”

“…The range of values probably reflects, on the one hand, the particular requirements for access of the footprinting reagent and, on the other, uncertainties as to the precise orientation of fluorescent or photoactivatable probes attached to DNA; again, the data are consistent with the proposed model but are insufficiently discriminating to rule out alternatives that could be proposed. Using time resolved fluorescence spectroscopy, it has been concluded that more of the duplex DNA upstream of the primer terminus is drawn into the binding site when the 3' terminus is at the 3'-5' exonuclease active site (60). The proposed model (19) suggests a difference of 2-3 basepairs between the polymerase and exonuclease binding modes; it does not appear able to accommodate the 9-nucleotide difference inferred from experiments that compared the effect of a bulky DNA substituent on the two enzymatic reactions (61).…”

“…It has been suggested (19) that the bent configuration modeled for duplex DNA bound to the polymerase site ( Figure 7) may serve to destabilize the duplex terminus and facilitate the melting necessary for the exonuclease reaction. Direct measurement by time-resolved fluores cence spectroscopy indicates that the energetics of melting is indeed in an appropriate range, since a perfectly basepaired duplex DNA molecule partitions about 7: 1 in favor of the polymerase site (60) . Protein sequence alignments suggest strongly that a very similar 3'-5' exonuclease active site will be found in all DNA polymerases that have an editing function, and preliminary structural data for the N-terminal 45-kDa domain of T4 DNA polymerase confirm this prediction (J Wang, P Yu, WH Konigsberg , TA Steitz, unpublished) .…”

Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69

“…In contrast, the interaction of the replicative DNA polymerase is, of necessity, confined to the primertemplate junction. The polymerase has a high affinity for this unique structure over either ssDNA or double-stranded DNA (4,5). The processivity of the polymerase is further enhanced by the sliding clamp family of proteins that tether the polymerase to the DNA (6,7).…”

Aptameric Molecular Switch for Cascade Signal Amplification

“…1B) and forming a 7-base duplex structure with itself that can initiate replication. The 15mer thrombin aptamer appears to have a dissociation constant (K d ) from 25 to 200 nmol/L for thrombin (26 ), whereas the Klenow fragment polymerase has a K d of 7.9 nmol/L for DNA (27 ). The thrombin aptamer has a greater K d for thrombin than the polymerase for DNA, so the polymerase is able to displace thrombin from the furled-up pocket on the 5Ј terminus.…”