Recognition of Sequence-Directed DNA Structure by the Klenow Fragment of DNA Polymerase I

Abstract

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.

Effects of DNA mismatches on binding affinity and kinetics of polymerase-DNA complexes as revealed by surface plasmon resonance biosensor

“…The K p?e increased by about 320-and 560-fold for DNA duplexes containing double (ATPM-5) and triple (ATPM-6) mismatches, respectively while the binding fraction at the exonuclease domain increased to > 93% for both mismatched DNA duplexes. The K p?e values and fractions of the various DNA bound at polymerase and exonuclease sites obtained from the SPR biosensor measurement are consistent with those obtained from the time-resolved fluorescence measurements, 23,24 thus further validating the utility of the biosensor as a sensitive technique to analyze complex binding kinetics. These previous results indicated that the presence of mismatch(es) within the DNA substrates results in greater occupancy of the 3A-5A exonuclease site of mutant enzyme.…”

Effect of the Oxidized Guanosine Lesions Spiroiminodihydantoin and Guanidinohydantoin on Proofreading by Escherichia coli DNA Polymerase I (Klenow Fragment) in Different Sequence Contexts

“…It should be noted that an Og:A mismatch placed in the GC-rich duplex (COgG/AC14) has approximately the same stability (∆∆G ) -0.6 ( 0.9 kcal/ mol) as the undamaged G:C-containing duplex (CGG/CC14), while the ∆∆G value for the AT-rich duplex is about -1.4 ( 0.2 kcal/mol (Table S1, Supporting Information). Thus, only the terminal Og:A mismatch in AT-rich sequence contexts is destabilizing enough to satisfy the free energy partitioning requirement calculated by Carver et al (32,33). Efficient excision of the Og:A mismatch in the GC-rich duplex cannot be explained by thermodynamic factors alone.…”

“…The free energy difference for partitioning of correct base pairs versus mismatches in undamaged DNA duplexes is found to be -0.6 to -0.7 kcal/mol, while the difference in melting free energies between correct base and mismatches is calculated to be -0.2 kcal/mol for blunt-ended duplexes (32,33). However, a much better correlation between 3′ editing and thermodynamic stability and the partitioning energy was observed by Morales and Kool (34) for oligonucleotides with overhanging ends.…”

Temperature Dependence and Thermodynamics of Klenow Polymerase Binding to Primed-Template DNA

“…Thus, there is little or no binding affinity dependence on the sequence of the single-stranded portion of this primedtemplate construct. Other slightly larger, but still quite subtle, sequence dependent effects have been observed previously for Klenow-DNA binding when the binding to different DNA constructs has been compared (21)(22)(23). None of these modest variations have indicated that Klenow displays any true sequence specificity, especially when compared to typical restriction endonucleases or transcriptional regulators.…”

“…DNA polymerases are generally considered essentially nonsequence-specific DNA binding proteins, although they do exhibit a limited amount of DNA sequence dependent variation in binding affinity (21)(22)(23). However, the span between the tightest and weakest binding sequences for a polymerase or other ''nonspecific'' DNA binding protein (e.g., E. coli SSB (24)) is generally on the order of 6 one order of magnitude: a range that is 10 3 -10 6 -fold smaller than the span between sequence-specific and nonspecific binding for a transcriptional regulator or a restriction endonuclease (18).…”