A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA

Abstract

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.