Kausiki Datta

Spectroscopic studies of position-specific DNA “breathing” fluctuations at replication forks and primer-template junctions

Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations (“DNA breathing”) can occur at these ssDNA–dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNA–dsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA–dsDNA junction. As expected, spectroscopically measurable unwinding penetrates ≈2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA–dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA–dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.

Direct Spectroscopic Study of Reconstituted Transcription Complexes Reveals That Intrinsic Termination Is Driven Primarily by Thermodynamic Destabilization of the Nucleic Acid Framework

Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, whereas the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-Aminopurine probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency, and disrupt the upstream end of the hybrid as well as interfere with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently “lock” the polymerase to the nucleic acid scaffold and, thus, hold the RNA-DNA hybrid “in frame.” At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, whereas hairpin-dependent pausing provides time for the process to go to completion.

DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3′-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary (“correct”) and noncomplementary (“incorrect”) NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3′-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

In addition to their capacity for template-directed 5′ → 3′ DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3′ → 5′ exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3′ terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.

Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids

We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base–base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA ‘breathing’ at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution.

Thermodynamics of the DNA Structural Selectivity of the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the Delta C(p) of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.

The Glutamate Effect on DNA Binding by Pol I DNA Polymerases: Osmotic Stress and the Effective Reversal of Salt Linkage

The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Deltan(ions)) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Deltan(ions) in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Deltan(ions). Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of approximately 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to approximately 150-200 waters released), suggesting that glutamate plays a significant osmotic role. Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.