Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.
DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.
Soultanas
P
P
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Dillingham
M S
MS
Wiley
P
P
Webb
M R
MR
Wigley
D B
DB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D010618:Phenanthrolines / Q000378:metabolism; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; D010618:Phenanthrolines; C084217:pcrA protein, Bacteria; C078638:copper-neocuproine complex; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D000251:Adenosine Triphosphatases; D004265:DNA Helicases