A SINGLE-STRANDED-DNA BINDING-PROTEIN BINDS THE ORIGIN OF REPLICATIONOF THE DUPLEX KINETOPLAST DNA

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatid s initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-st randed DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) doe s not bind a duplex UMS dodecamer, it binds the double-stranded kDNA m inicircle as well as a duplex minicircle fragment containing the origi n-associated UMS. Binding of the minicircle origin region by the singl e-stranded DNA binding protein suggested the local unwinding of the DN A double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or other wise sharply distorted DNA at the minicircle origin region. Computer a nalysis predicts the sequence-directed curving of the minicircle origi n region. Electrophoresis of a minicircle fragment containing the orig in region in polyacrylamide gels revealed a significantly lower electr ophoretic mobility than expected from its length. The fragment anomalo us electrophoretic mobility is displayed only in its native conformati on and is dependent on temperature and gel porosity, indicating the lo cal curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwi nding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstac ked dinucleotide sequences at the bent origin site.