Open AccessPurification and Characterization of a Mitochondrial, Single‐Stranded‐DNA–Binding Protein from Paracentrotus Lividus Eggs
Author(s) -
Roberti Marina,
Musicco Clara,
Loguercio Polosa Paola,
Gadaleta Maria Nicola,
Quagliariello Ernesto,
Cantatore Palmiro
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00052.x
Subject(s) - paracentrotus lividus , cooperativity , tetramer , biology , dna , cooperative binding , mitochondrial dna , biochemistry , polynucleotide , oligonucleotide , dna binding protein , biophysics , microbiology and biotechnology , binding site , sea urchin , enzyme , gene , transcription factor
A binding protein for single‐stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE‐Sephacel and single‐stranded‐DNA–cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single‐stranded‐DNA–binding protein exists as a homo‐oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single‐stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding‐site size, determined by gel mobility‐shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single‐stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2×10 9 M −1 , a value which is the highest among the mitochondrial single‐stranded‐DNA–binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.