title: Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein Integrase Interactor 1 (INI1/hSNF5).
abstract: Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105-183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K'd1 = 222 µM and K'd2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = -29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH'1 = 115.7 KJ/mole, TΔS'1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH'2 = -106.3 KJ/mole, TΔS'2 = -75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1.
authors.affiliation: Department of Biochemistry, University of Calcutta, Kolkata, India. radas_03@yahoo.com
authors.forename: Supratik
authors.initials: S
authors.lastname: Das
authors.forename: Baisakhi
authors.initials: B
authors.lastname: Banerjee
authors.forename: Maidul
authors.initials: M
authors.lastname: Hossain
authors.forename: Muruganandan
authors.initials: M
authors.lastname: Thangamuniyandi
authors.forename: Saumya
authors.initials: S
authors.lastname: Dasgupta
authors.forename: Nipa
authors.initials: N
authors.lastname: Chongdar
authors.forename: Gopinatha Suresh
authors.initials: GS
authors.lastname: Kumar
authors.forename: Gautam
authors.initials: G
authors.lastname: Basu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010944:Plants / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071796:SMARCB1 Protein; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; D014157:Transcription Factors; D004247:DNA; D019427:HIV Integrase