Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. 2003

Rodrigo A Villanueva, and Colleen B Jonsson, and Jennifer Jones, and Millie M Georgiadis, and Monica J Roth
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni(2+)-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008979 Moloney murine leukemia virus A strain of Murine leukemia virus (LEUKEMIA VIRUS, MURINE) arising during the propagation of S37 mouse sarcoma, and causing lymphoid leukemia in mice. It also infects rats and newborn hamsters. It is apparently transmitted to embryos in utero and to newborns through mother's milk. Moloney Leukemia Virus,Leukemia Virus, Moloney,Virus, Moloney Leukemia
D011489 Protein Denaturation Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein. Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D016662 Virus Integration Insertion of viral DNA into host-cell DNA. This includes integration of phage DNA into bacterial DNA; (LYSOGENY); to form a PROPHAGE or integration of retroviral DNA into cellular DNA to form a PROVIRUS. Integration, Provirus,Integration, Virus,Provirus Integration,Viral integration,Integrations, Provirus,Integrations, Virus,Provirus Integrations,Viral integrations,Virus Integrations,integration, Viral,integrations, Viral
D017354 Point Mutation A mutation caused by the substitution of one nucleotide for another. This results in the DNA molecule having a change in a single base pair. Mutation, Point,Mutations, Point,Point Mutations
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus
D019281 Dimerization The process by which two molecules of the same chemical composition form a condensation product or polymer. Dimerizations
D019426 Integrases Recombinases that insert exogenous DNA into the host genome. Examples include proteins encoded by the POL GENE of RETROVIRIDAE and also by temperate BACTERIOPHAGES, the best known being BACTERIOPHAGE LAMBDA. Integrase

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