Abstract
Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5'-GGGGTTGGTGTA-3'). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not double-stranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.
Highlights
Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5-GGGGTTGGTGTA-3)
A specific originbinding protein that interacts with the origin-associated UMS, is a likely candidate to function in the process of replication initiation and may play a role in a mechanism that coordinates Kinetoplast DNA (kDNA) minicircle and maxicircle replication
It is within this context that we had searched for and isolated a UMS-binding protein from C. fasciculata cell extracts
Summary
Nucleic Acids, Nucleotides, Proteins, and Resins—Synthetic deoxyoligonucleotides were prepared by an Applied Biosystems oligonucleotide synthesizer at the Bletterman Laboratory of the Interdepartmental Division, Faculty of Medicine, the Hebrew University of Jerusalem. One unit of UMSBP is defined as the amount of protein required for binding of 1 fmol of UMS H-strand DNA probe under the standard mobility-shift assay conditions [17]. 155-l fractions were collected dropwise from the bottom of the tube and assayed for the presence of the different proteins as follows: UMSBP was assayed by the standard mobility-shift assay; cytochrome C and hemoglobin following A405; DNA polymerase I by nick-translation assay modified from the method of Richardson et al [30]; and peroxidase activity following the increase at A405 resulted from the oxidation of pyrogallol to purpurogallin, as recommended by the manufacturer (Sigma). Experiments were carried out under the standard mobility-shift assay conditions, by serial dilution of both UMSBP and the oligonucleotide probe while keeping their molar ratios constant. Protein Sequence Analysis—Protein sequences were analyzed using the Genetics Computer Group software package (1991), Madison, Wisconsin
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