title: Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates.
abstract: The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNA-protein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time.
authors: Department of Cell and Molecular Biology, University of Uppsala and Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Pernilla
authors: P
authors: Lagerbäck
authors: Evalena
authors: E
authors: Andersson
authors: Christer
authors: C
authors: Malmberg
authors: Karin
authors: K
authors: Carlson
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D004247:DNA; D003850:Deoxyribonuclease I