ABSTRACT
Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages. In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins. We have analysed the structural properties of the different protein-DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy. Analysis of the UvrA(2)B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns. In the UvrB-DNA pre-incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding. Based on these results, a role for DNA wrapping in damage recognition is proposed. Evidence is presented that DNA wrapping in the pre-incision complex also stimulates the rate of incision by UvrC.
Subject(s)
DNA Helicases/metabolism , DNA Repair , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Endodeoxyribonucleases , Escherichia coli Proteins , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , DNA Helicases/chemistry , DNA Primers/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Macromolecular Substances , Microscopy, Atomic Force , Molecular Sequence DataABSTRACT
Nucleotide excision repair in Escherichia coli is a multistep process in which DNA damage is removed by incision of the DNA on both sides of the damage, followed by removal of the oligonucleotide containing the lesion. The two incision reactions take place in a complex of damaged DNA with UvrB and UvrC. It has been shown (Lin, J. -J., and Sancar, A. (1992) J. Biol. Chem. 267, 17688-17692) that the catalytic site for incision on the 5' side of the damage is located in the UvrC protein. Here we show that the catalytic site for incision on the 3' side is in this protein as well, because substitution R42A abolishes 3' incision, whereas formation of the UvrBC-DNA complex and the 5' incision reaction are unaffected. Arg(42) is part of a region that is homologous to the catalytic domain of the homing endonuclease I-TevI. We propose that the UvrC protein consists of two functional parts, with the N-terminal half for the 3' incision reaction and the C-terminal half containing all the determinants for the 5' incision reaction.
Subject(s)
Bacterial Proteins/metabolism , Catalytic Domain , DNA Repair , Endodeoxyribonucleases , Escherichia coli/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , DNA, Bacterial , Escherichia coli Proteins , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Three new cholesterol-containing phosphoramidites where synthesized and used in automated synthesis of modified DNA fragments. These cholesterol lesions are good substrates for the E. coli UvrABC endonuclease. In vitro they are incised from damaged DNA with higher efficiency in respect with the cholesterol lesions previously published.
