Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplastDNA minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H)) strand in itiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is con served in the minicircles of trypanosomatid species. A protein from the try panosomatid Crithidia fasciculata binds specifically a 14-mer sequence, con sisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin, This protein was identified as the prev iously described universal minicircle sequence (UMS)-binding protein (UMSBP ) (Tzfati, Y,, Abeliovich, H,, Avrahami, D,, and Shlomai, J, (1995) J. Biol . Chem, 270, 21339-21345), This CCHC-type zinc finger protein binds the sin gle-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the rep lication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protei n relies on their co-purification from C, fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequenc es. Both the conserved H-strand hexamer and its nanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes, Stoichiometry analysis indi cates that each UMSBP molecule binds either of the two origin-associated se quences in the nucleoprotein complex but not both simultaneously.