tailieunhanh - Báo cáo khoa học: Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro

Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cellDNA. TheC-terminal domainof INis involved in DNA binding and enzyme multimerization. We previ-ously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma vir-uses (ALSV) IN [Moreauet al. (2002). Arch. , 1761–1778]. Here, we modelled these IN mutants and ana-lysed their ability tomediate concertedDNA integration (in anin vitroassay) as well as to formdimers (by size exclusion chromatography and protein–protein cross-linking). . | Eur. J. Biochem. 270 4426-4438 2003 FEBS 2003 doi Mutations in the C-terminal domain of ALSV Avian Leukemia and Sarcoma Viruses integrase alter the concerted DNA integration process in vitro Karen Moreau1 Claudine Faure1 Sébastien Violot2 3 Gerard Verdier1 3 and Corinne Ronfort1 3 1 Universite Claude Bernard Centre National de la Recherche Scientifique Institut National de la Recherche Agronomique Lyon France 2Institut de Biologie et Chimie des Proteines Centre National de la Recherche Scientfique Laboratoire de Bio-Cristallographie Universite Claude Bernard France 3IFR 128 BioSciences Lyon Gerland Lyon France Integrase IN is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses ALSV IN Moreau et al. 2002 . Arch. Virol. 147 1761-1778 . Here we modelled these IN mutants and analysed their ability to mediate concerted DNA integration in an in vitro assay as well as to form dimers by size exclusion chromatography and protein-protein cross-linking . Mutations of residues located at the dimer interface V239 L240 Y246 V257 and K266 have the greatest effects on the activity of the IN. Among them a the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization b the V239A V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations V260E and Y246W AC25 highlight the role of the C-terminal domain in the general folding of the enzyme and hence on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted