Function of the homologous regions of the Escherichia coli DNA excision repair proteins UvrB and UvrC in stabilization of the UvrBC-DNA complex and in 3'-incision.
The nicking of damaged DNA during the nucleotide excision repair reaction in E. coli, is the result of a multi-step process involving three enzymes, UvrA, UvrB and UvrC. The UvrB protein is loaded on the site of the damage by UvrA, forming a stable UvrB-DNA complex. This complex is recognized by UvrC and in the resulting UvrBC-DNA complex dual incision takes place, first on the 3'-side and next on the 5'-side of the damaged nucleotide. A domain in the C-terminal part of UvrB has been identified to be essential for formation of the specific UvrBC-DNA complex that induces the 3'-incision [1]. The N-terminal half of UvrC contains a region that is homologous to this C-terminal domain of UvrB. Using site-directed mutagenesis of a conserved phenylalanine in the homologous regions of UvrB and UvrC two mutants were constructed, UvrB(F652L) and UvrC(F223L). Both proteins were tested in vitro using a DNA substrate with a defined cisplatin lesion. The protein-DNA and protein-protein interactions were studied using bandshift assays and DNAse I footprinting. We show that both domains are important for the binding of UvrC to the UvrB-DNA complex.
Moolenaar
G F
GF
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Franken
K L
KL
van de Putte
P
P
Goosen
N
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004265:DNA Helicases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D010957:Plasmids; D011485:Protein Binding; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C066567:UvrC protein, E coli; D004265:DNA Helicases