Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature

Shatzky-Schwartz, Michal; Hiller, Y; Reich, Ziv; Ghirlando, Rodolfo; Weinberger, Sarah; Minsky, Abraham

doi:10.1021/bi00123a019

Cited by 12 publications

(6 citation statements)

References 47 publications

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“…This result is consistent with the greater rate of exonucleolysis seen for (A+T)-rich sequences compared with (G+C)-rich sequences (22). In addition to increasing the melting capacity ofthe primer terminus, homopolymeric A/T runs promote primer-template slippage under certain conditions and may have an altered DNA conformation at the primer terminus that inhibits DNA binding at the polymerase site (23,24).…”

Section: Discussionsupporting

confidence: 61%

Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

Carver

Hochstrasser

Millar

1994

Proc. Natl. Acad. Sci. U.S.A.

123

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Fluorescence depolarization decays were measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer27-mer oligonucleotides bound to Klenow framet of DNA polymerase I. A necessary feature of all DNA-dependent DNA polymerases is the ability to copy DNA with high fidelity; for DNA polymerase I, the probability of an error during replication is <10-6 per nt addition (1). Exonucleolytic proofreading contributes to replication fidelity through the preferential excision of mismatched bases after mispair synthesis occurs (for reviews, see refs. 2-4). In DNA polymerase I, proofreading is accomplished by a 3'-5' exonuclease in a separate domain of the same polypeptide with 5'-3' polymerase activity. The crystal structure of the 70-kDa Klenow fragment of DNA polymerase I has been determined. Klenow fragment consists of two putative DNA-binding clefts divided by a "thumb-like" domain (5). The 5'-3' polymerase and 3'-5' exonuclease activities are separated by [30][31][32][33][34][35][36][37][38][39][40] A, indicating that shuttling of the DNA between the two sites is a requirement for proofreading. Joyce and Steitz (6) have proposed a structural model for movement of DNA from the polymerase site to the exonuclease site. In this model, 3-to 4-bp melt at the primer terminus, followed by movement of the resulting single-stranded region of DNA into the exonuclease site (7). From assays using biotinylated DNA substrates, there is evidence that the polymerase-exonuclease transition involves an 8-bp translocation of the protein upstream on the DNA (8). However, a recent structure of duplex DNA bound to Klenow fragment shows the primer terminus bound in the cleft adjacent to the exonuclease site, much closer to both active sites than thought (9). Currently, the mechanism of DNA movement within the protein remains unclear. In addition, the effects of protein and DNA structure upon the binding of DNA to each site have not been resolved.The kinetics of misincorporation and proofreading in the Klenow fragment have been examined in detail using quenched-flow techniques, and the scheme required to describe interconversion of the intermediate states is complex (10,11). For a simple model in which only DNA binding is considered, preferential excision of mismatched bases is achieved by increasing the rate of exonucleolysis of the terminal nucleotide relative to the rate of addition of the next nucleotide. An increase in the ratio of exonuclease to polymerase rates could be due to a decrease in the intrinsic rate of polymerization, kpol, or to an increase in the partitioning of DNA from the polymerase site into the exonuclease site, or to a combination of these effects (Fig. 1). The greater melting capacity of mispaired termini could contribute to increased partitioning of DNA into the exonuclease site (12). In addition, it is important to note that any structural feature of the DNA favoring DNA binding at the exonuclease site over binding at the polymerase site will increase the rate of exonucleo...

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Section: Discussionsupporting

confidence: 61%

Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

Carver

Hochstrasser

Millar

1994

Proc. Natl. Acad. Sci. U.S.A.

123

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“…These effects were explained by assuming that the enzyme prefers a DNA substrate with a minor groove of average width. Shatzky-Schwartz et al (13) have shown that the activities of several DNA-processing enzymes, including DNase I and the exonucleases BAL31 and exoIII, are markedly impaired by the presence of repeated A-tract sequences (5′-A 4 T 4 -3′) within their duplex DNA substrates. These effects were attributed to a reduced binding affinity of the enzymes for bent regions of DNA.…”

Section: Discussionmentioning

confidence: 99%

“…Investigations of DNA structure during the past decade have shown that significant sequence-dependent deviations from the canonical B-form helical structure can occur. These differences, although small relative to differences between the "A", "B," and "Z" DNA structures, can have a pronounced effect upon sequence-independent enzymes that use duplex DNA as a substrate (13,14). Sequence-directed DNA curvature is correlated with a variety of protein-DNA interactions in vivo, and DNA bending may be important in sequence recognition by DNA-binding proteins (15).…”

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confidence: 99%

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

Carver

Millar

1998

Biochemistry

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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.

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“…In addition, it was recently reported that DNA-protein interactions can be attenuated by the presence of intrinsic sequence-dependent DNA curvature (Shatzky-Schwarz et al, 1992). It is interesting to speculate whether a (+)-CC-1065-entrapped or -induced bent conformation of DNA might mimic an intrinsic or proteininduced curved DNA found in the regulatory regions of a genome, such as the promoter regions, or in replication origins (Le,, serve as a surrogate protein).…”

Section: Discussionmentioning

confidence: 99%

A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR

Sun¹,

Lin²,

Hurley³

1993

Biochemistry

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(+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol, 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3' adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3' side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5'-AGTTA* or 5'-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5'-AGTTA* the bending magnitude increases with increased temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

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Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature

Cited by 12 publications

References 47 publications

Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

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

A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR

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