Kinetoplast DNA (kDNA) 1 is a unique extrachromosomal DNA network found in the single mitochondrion of parasitic flagellated protozoa of the family Trypanosomatidae. In Crithidia fasciculata, kDNA consists of about 5,000 DNA minicircles (2.5 kilobase pairs each) and about 50 DNA maxicircles (37 kilobase pairs each) interlocked topologically to form a DNA network (reviewed in Refs. 1-7). Minicircles, in most trypanosomatid species, are heterogeneous in sequence. However, two short sequences that are associated with the process of replication initiation and are located 70 -100 nucleotides apart on the minicircle complementary DNA strands were conserved in minicircles of all the trypanosomatid species studied: the sequence GGGGTTGGTGTA, known as the universal minicircle sequence (UMS), in the minicircle heavy (H) strand, and the sequence ACGCCC in its light (L) strand (3).The replication of kDNA is restricted to the discrete S-phase, approximately in parallel with the replication of the nuclear DNA (reviewed in Refs. 4,5,8,and 9). According to the current model for the network replication (10, 11), during the S-phase individual minicircles are released from the central zone of the network and replicate, each forming two nicked (and gapped) progeny minicircles that reattach to the periphery of the network (10 -14). At the end of the S-phase, the network is composed of replicated, nicked, and gapped circles and has doubled in size. The final steps, which occur at the beginning of the G 2 phase, include the physical splitting of the double-size network and the covalent closure of the circles, followed by the network segregation during cell division. Immunolocalization and in situ hybridization studies have co-localized free replicating minicircles and replication proteins to two peripheral antipodal sites of the kinetoplast disc, suggesting that these are the sites in which minicircle replication is conducted through the action of two replication complexes (14 -16).The replication of free minicircles has been studied in Trypanosoma equiperdum (17-20), C. fasciculata (21-25), and Leishmania tarentolae (26,27). Minicircle replication initiates by the synthesis of an RNA primer at the UMS site. Elongation of the nascent L-strand displaces the parental L-strand, which serves as a template for the discontinuous H-strand synthesis.We have previously reported on the presence in C. fasciculata of a sequence-specific single-stranded DNA-binding protein that binds specifically to the UMS. The protein, designated UMSBP, was purified to apparent homogeneity from C. fasciculata cell extracts (28,29), and its encoding gene was cloned and analyzed (30,31). The UMS-binding protein is a CCHCtype zinc finger dimer protein (31) of 27.4 kDa with a 13.7-kDa protomer (28). Whereas neither the duplex form of a UMS dodecamer nor a quadruplex DNA conformation are bound by UMSBP, the protein binds efficiently the natural doublestranded kDNA minicircle, as well as a duplex minicircle fragment containing the origin-associated UMS (32). These s...