DNA-induced dimerization of the Escherichia coli Rep helicase.
The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 microM (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-gamma-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 less than or equal to n less than or equal to 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n less than or equal to 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.
Chao
K L
KL
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843-2128.
Lohman
T M
TM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D003090:Coliphages / Q000235:genetics; D004265:DNA Helicases / Q000187:drug effects / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C506744:rep protein, E coli; D004120:Dimethyl Suberimidate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases