title: Photocaged variants of the MunI and PvuII restriction enzymes.
abstract: Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Silanskas
authors.forename: Michael
authors.initials: M
authors.lastname: Foss
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wende
authors.forename: Claus
authors.initials: C
authors.lastname: Urbanke
authors.forename: Arunas
authors.initials: A
authors.lastname: Lagunavicius
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D015077:2-Hydroxy-5-nitrobenzyl Bromide / Q000737:chemistry; D001483:Base Sequence; D055162:Biocatalysis / Q000528:radiation effects; D002942:Circular Dichroism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000528:radiation effects; D008958:Models, Molecular; D015394:Molecular Structure; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D009841:Oligonucleotides; D015077:2-Hydroxy-5-nitrobenzyl Bromide; C059776:endodeoxyribonuclease MfeI; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific