RecA-mediated cleavage of lambda cI repressor accepts repressor dimers: probable role of prolyl cis-trans isomerization and catalytic involvement of H163, K177, and K232 of RecA.
The lambda cI repressor is found to be cleaved in the presence of activated RecA in its DNA-bound dimeric form at a rate similar to that in the absence of operator DNA in contrast to previous studies inferring repressor monomer as a preferred substrate. Though activated RecA does not possess any measurable isomerase activity against a standard peptide substrate, prolyl isomerase inhibitors cyclosporin A and rapamycin do inhibit RecA-mediated cleavage. Histidine and lysine to a smaller extent, are shown to cleave cI repressor in a non-enzymatic fashion whereas arginine and glutamate do not. When activated RecA filament is covalently modified by using an excess of diethyl pyrocarbonate or maleic anhydride, RecA-mediated cleavage of cI repressor is inhibited. Combining our chemical modification data with model building and earlier mutagenesis data, it is argued that H163, K177, and K232 in RecA are crucial residues involved in cI repressor cleavage by combining with the catalytic Ser149 and K192 in the repressor. It is suggested by model building that subunits n, n+4, and n+5 in the RecA filament contribute one loop each for holding the C-terminal domain of the repressor during cleavage within the RecA helical groove, explaining why its ADP-form is inactive and its ATP-form is active regarding repressor cleavage.
Pal
Atasi
A
Department of Biochemistry, Bose Institute P-1/12, C.I.T. Scheme VIIM Calcutta 700054, India.
Chattopadhyaya
Rajagopal
R
D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D008958:Models, Molecular; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D011938:Rec A Recombinases; D019696:Peptidylprolyl Isomerase