Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement
AUTOR(ES)
Firman, Keith
FONTE
Oxford University Press
RESUMO
The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, the data can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can ‘reset’ in either direction whenever the DNA is released.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=305691Documentos Relacionados
- Repercussions of DNA tracking by the type IC restriction endonuclease EcoR124I on linear, circular and catenated substrates.
- Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I.
- Non-specific binding of restriction endonuclease EcoR1 to DNA.
- Purification and biochemical characterisation of the EcoR124 type I modification methylase.
- A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity.
