Genetic identification of the DNA binding domain of Escherichia coli LexA protein.

Abstract The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in ~40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of ~10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

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Purified estrogen receptor DNA binding domain expressed in Escherichia coli activates transcription of an estrogen-responsive promoter in cultured cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)54394-5 ◽

1991 ◽

Vol 266 (35) ◽

pp. 24070-24076 ◽

Cited By ~ 2

Author(s):

A.M. Nardulli ◽

D. Lew ◽

L. Erijman ◽

D.J. Shapiro

Keyword(s):

Escherichia Coli ◽

Estrogen Receptor ◽

Dna Binding ◽

Cultured Cells ◽

Binding Domain ◽

Dna Binding Domain

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The Identification of the Single-Stranded DNA-Binding Domain of the Escherichia coli RecA Protein

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.419_2.x ◽

1995 ◽

Vol 233 (2) ◽

pp. 419-425 ◽

Cited By ~ 22

Author(s):

Renee V. Gardner ◽

Oleg N. Voloshin ◽

R. Daniel Camerini-Otero

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Reca Protein ◽

Binding Domain ◽

Dna Binding Domain ◽

Single Stranded Dna

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Crystallization and Low Temperature Diffraction Studies of the DNA Binding Domain of the Single-stranded DNA Binding Protein from Escherichia coli

Journal of Molecular Biology ◽

10.1006/jmbi.1994.1453 ◽

1994 ◽

Vol 240 (4) ◽

pp. 396-399 ◽

Cited By ~ 8

Author(s):

Jennifer M. Thorn ◽

Paul D. Carr ◽

John W. Chase ◽

Nicholas E. Dixon ◽

David L. Ollis

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Low Temperature ◽

Binding Protein ◽

Dna Binding Protein ◽

Binding Domain ◽

Dna Binding Domain ◽

Single Stranded Dna

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Specific Contacts between Residues in the DNA-Binding Domain of the TyrR Protein and Bases in the Operator of thetyrP Gene of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.8.2338-2345.1999 ◽

1999 ◽

Vol 181 (8) ◽

pp. 2338-2345 ◽

Cited By ~ 7

Author(s):

J. S. Hwang ◽

J. Yang ◽

A. J. Pittard

Keyword(s):

Escherichia Coli ◽

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Binding Affinities ◽

Wild Type ◽

Small Reduction ◽

Gel Shift Assay ◽

Gel Shift

ABSTRACT In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051–1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the ±5 positions in the TyrR boxes.

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