Protein-DNA interactions and alterations in the DNA structure upon UvrB-DNA preincision complex formation during nucleotide excision repair in Escherichia coli.
The UvrB-DNA preincision complex is a key intermediate in the repair of damaged DNA by the UvrABC endonuclease from Escherichia coli. DNaseI footprinting of this complex on DNA with a cis-[Pt(NH3)2[d(GpG)-N7(1),N7(2)]] adduct provided global information on the protein binding site on this substrate [Visse, R., et al. (1991) J. Biol. Chem. 266, 7609-7617]. By applying a method developed by Fairall and Rhodes [Fairall, L., & Rhodes, D. (1992) Nucleic Acids Res. 20, 4727-4731], who have used the size and shape of DNasI for the interpretation of a footprint, we were able to define in more detail the region where UvrB-DNA interactions in the preincision complex occur. The potential interactions with phosphate groups could be reduced to less then 14 in the damaged and to 12 in the nondamaged strand. The main UvrB-DNA interactions seem restricted to the major groove on both sides of the lesion. As a consequence UvrB crosses the minor groove just downstream of the damage. Such a binding of UvrB orients the protein away from the damage. The more detailed interpretation of UvrB-DNA interactions was supported by methylation protection experiments. The structure of the DNA in the preincision complex formed on cis-[Pt(NH3)2[GpG-N7(1),N7(2)]] is altered as could be shown diethylpyrocarbonate sensitivity of adenines just downstream of the lesion. However the adenines just downstream of another cisplatin adduct, cis-[Pt(NH3)2[d(GpCpG)-N7(1),N7(3)]], did not become diethylpyrocarbonate sensitive in the preincision complex although this complex is incision proficient.(ABSTRACT TRUNCATED AT 250 WORDS)
Visse
R
R
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
King
A
A
Moolenaar
G F
GF
Goosen
N
N
van de Putte
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D011196:Potassium Permanganate; D013329:Structure-Activity Relationship; D013941:Thymine / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D011196:Potassium Permanganate; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C066567:UvrC protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004047:Diethyl Pyrocarbonate; D013941:Thymine