Recognition Mechanisms of DNA-Specific Enzymes

Jovin, T. M.

doi:10.1146/annurev.bi.45.070176.004325

Cited by 70 publications

(25 citation statements)

References 122 publications

(184 reference statements)

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“…DNA-protein interactions are of fundamental importance in a large variety of molecular processes, yet are not well understood biochemically (1,2). We are currently studying the lac operator-repressor system in order to determine the mechanisms by which proteins recognize and interact with specific DNA sequences.…”

mentioning

confidence: 99%

How lac repressor recognizes lac operator.

Goeddel¹,

Yansura²,

Caruthers³

1978

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

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Nucleotide analogs were substituted for unmodified nucleotides at specific sites in the lac operator sequence by a combination of chemical and enzymatic procedures. The nitrocellulose filter assay was used to-study the interactions of these modified operators with wild-type (SQ) and tight-binding (QX86) lac repressors. These studies iicat directly the 5 methyl of thymine and the 2 amino of ganine as important operator-repressor contact sites. Furthermore, when these findings are combined with published results from other laboratories, a model for the lac operator-Iac repressor interaction can be derived. Two important postulates follow from this model. (il The repressor interacts at specific and defined sites with the N7 of fuanine, the 5 methyl of thymine, the 2 amino of guanine, and the central major groove of the operator.(ii) The repressor binds to one side of the operator. DNA-protein interactions are of fundamental importance in a large variety of molecular processes, yet are not well understood biochemically (1,2). We are currently studying the lac operator-repressor system in order to determine the mechanisms by which proteins recognize and interact with specific DNA sequences.The lac repressor protein binds tightly to the lac operator, a unique sequence in the Escherichia coli chromosome. An examination of operator-constitutive )Oc) mutants has identified eight base pairs that are involved in binding to repressor (8. 4). Gilbert et al. (5) have shown that the binding of repressor to operator specifically protects four guanines and three adenines against methylation with dimethyl sulfate. At the same time, the methylation of two guanines and one adenine is enhanced. (Dimethyl sulfate methylates double-stranded DNA at the N7 of guanine and the N3 of adenine exposed, respectively, in the major and minor grooves.) Crosslinking experiments have identified thymine residues that contact repressor in the major groove (6). Thus the lac repressor appears to interact with operator DNA in both the major and minor grooves. All these sets of data are shown in Fig. 1.One primary objective of research from this laboratory is to decipher those elempts of the lac operator bases that stabilize the repressor-operator (RO) complex. The approach outlined in this paper involves insertion of nucleic acid base analogs at specific sites in the DNA, followed by analysis of how these analogs affect the stability of the RO interaction. These results, when used in conjunction with the above sets of data, have allowed us to deduce several lac operator sites that are recognized by lac repressor.MATERIALS AND METHODS Syntheses of unmodified and various modified lac operator DNAs have either been described (7-11) or remain to be published. Nitrocellulose filter binding assays were performed as described previously (9,12,13). Wild-type (SQ) repressor was purified by a published procedure (9). Tight-binding (QX86) repressor was provided by J. Sadler. RESULTSThe 26 operator duplexes listed in Table 1 were prepared by a combination...

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mentioning

confidence: 99%

How lac repressor recognizes lac operator.

Goeddel¹,

Yansura²,

Caruthers³

1978

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

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“…Open stretches of an otherwise double-helical DNA structure may always be present for very short periods of time as suggested by v. Hippel and McGhee [17]. Confirmatory data for the existence of these single-stranded structures have been presented recently; for references see [24]. To these structures tyrocidine could bind and would prevent reannealing.…”

Section: Discussionmentioning

confidence: 84%

The DNA · Tyrocidine Complex and Its Dissociation in the Presence of Gramicidin D

Chakraborty

Hansen

Ristow

et al. 1978

European Journal of Biochemistry

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The peptide antibiotic tyrocidine affects transcription in vitro by interfering with the initiation process. The complex formed between tyrocidine and native DNA is stable against nucleolytic enzymes. It is sensitive to variations in the salt concentration. Protection of DNA as a function of the tyrocidine concentration follows a curve suggesting cooperative binding of tyrocidine to DNA. The complex formation at low tyrocidine concentrations is accompanied by the presence of single stranded regions of unknown length. With single-stranded DNA, tyrocidine forms a complex which is relatively insensitive to high salt concentrations. Both native and single-stranded DNAs of the complexes become digestible in the presence of gramicidin D, another peptide antibiotic. Breakdown of the complex as a function of the gramicidin D concentration follows a curve which is the reverse of cooperative binding. Gramicidin D acts more effectively on single-stranded rather than on double-stranded DNA . tyrocidine complexes. The DNA . tyrocidine complex, which is broken down by formamide and dimethyl sulfoxide is stabilized by gramicidin D. The implications of these results are discussed with respect to a possible recognition of DNA sequences by tyrocidine and the interaction between DNA, tyrocidine and gramicidin D.

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“…Contrary to holo enzyme (fig2B) the core enzyme is less reactive and the C' fixation in figAA shifts to higher antigen concentrations on ~cubation with poly [d(A-T)] at 37°C. Molecular details of binding and recognition of DNA bindiig enzymes are still not fully explained, although conformational changes in the protein on binding to DNA have been suggested [7]. There is no direct approach at hand (such as X-ray crystallography) to investigate the molecular basis of conformational 2) with holo enzyme.…”

Section: Resultsmentioning

confidence: 99%

Conformational changes of Escherichia coli RNA polymerase on binding of templates

Stender¹

1979

FEBS Letters

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Recognition Mechanisms of DNA-Specific Enzymes

Cited by 70 publications

References 122 publications

How lac repressor recognizes lac operator.

How lac repressor recognizes lac operator.

The DNA · Tyrocidine Complex and Its Dissociation in the Presence of Gramicidin D

Conformational changes of Escherichia coli RNA polymerase on binding of templates

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