The RecBCD enzyme initiation complex for DNA unwinding: enzyme positioning and DNA opening.
The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an initiation complex for DNA unwinding. We studied the structure of these complexes formed with blunt-ended, 5'-extended, and 3'-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg(2+)-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5' end were only partially reactive to KMnO4, suggesting that part of the 5'-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.
Farah
J A
JA
Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
Smith
G R
GR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006834:Hydrazines / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000378:metabolism
D004277:DNA, Single-Stranded; D006834:Hydrazines; D013463:Sulfuric Acid Esters; D011196:Potassium Permanganate; C029424:hydrazine; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D003850:Deoxyribonuclease I; D004265:DNA Helicases; D008274:Magnesium; C007482:dimethyl sulfate