PURIFICATION AND CHARACTERIZATION OF A MITOCHONDRIAL, SINGLE-STRANDED-DNA-BINDING PROTEIN FROM PARACENTROTUS-LIVIDUS EGGS

A binding protein for single-stranded DNA was purified from Paracentro tus 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 sedimentati on analysis suggested that P. lividus mitochondrial single-stranded-DN A-binding protein exists as a homo-oligomer, possibly a tetramer, in s olution, The protein shows a stronger preference for poly(dT) with res pect to single-stranded M13, poly(dI) and poly(dC). Binding to poly(da ) takes place with much lower affinity, The binding-site size, determi ned by gel mobility-shift experiments with oligonucleotides of differe nt length, is approximately 45 nucleotides. The binding to single-stra nded 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 2x10(9) M-1, a value whic h is the highest among the mitochondrial single-stranded-DNA-binding p roteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein an d might be related to the peculiar mechanism of sea urchin mitochondri al DNA replication.