Thymidine Glycol: The Effect on DNA Structure and DNA Binding by Site-Specific Proteins

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

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Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.6.4.1218-1227.1986 ◽

1986 ◽

Vol 6 (4) ◽

pp. 1218-1227

Author(s):

L Naumovski ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Excision Repair ◽

Random Mutagenesis ◽

Nucleotide Binding ◽

Yeast Cells ◽

Wild Type ◽

Binding Region ◽

Site Specific ◽

Essential Function

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.

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Site-specific DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein.

The EMBO Journal ◽

10.1002/j.1460-2075.1985.tb03783.x ◽

1985 ◽

Vol 4 (5) ◽

pp. 1345-1349 ◽

Cited By ~ 27

Author(s):

J.R. Greene ◽

E.P. Geiduschek

Keyword(s):

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Type Ii ◽

Site Specific

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Borrelia burgdorferi EbfC, a Novel, Chromosomally Encoded Protein, Binds Specific DNA Sequences Adjacent to erp Loci on the Spirochete's Resident cp32 Prophages

Journal of Bacteriology ◽

10.1128/jb.00005-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4331-4339 ◽

Cited By ~ 25

Author(s):

Kelly Babb ◽

Tomasz Bykowski ◽

Sean P. Riley ◽

M. Clarke Miller ◽

Edward DeMoll ◽

...

Keyword(s):

Dna Binding ◽

Borrelia Burgdorferi ◽

Dna Sequences ◽

Membrane Surface ◽

Factor H ◽

Host Protein ◽

Site Specific ◽

Isolation And Characterization ◽

Wide Range ◽

Complement Regulator

ABSTRACT All examined isolates of the Lyme disease spirochete, Borrelia burgdorferi, naturally maintain numerous variants of a prophage family as circular cp32 episomes. Each cp32 carries a locus encoding one or two different Erp outer membrane, surface-exposed lipoproteins. Many of the Erp proteins bind a host complement regulator, factor H, which is hypothesized to protect the spirochete from complement-mediated killing. We now describe the isolation and characterization of a novel, chromosomally encoded protein, EbfC, that binds specific DNA sequences located immediately 5′ of all erp loci. This is one of the first site-specific DNA-binding proteins to be identified in any spirochete. The location of the ebfC gene on the B. burgdorferi chromosome suggests that the cp32 prophages have evolved to use this bacterial host protein for their own benefit and that EbfC probably plays additional roles in the bacterium. A wide range of other bacteria encode homologs of EbfC, none of which have been well characterized, so demonstration that B. burgdorferi EbfC is a site-specific DNA-binding protein has broad implications across the eubacterial kingdom.

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