Abstract
Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously 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 single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking 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 indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.
Highlights
Kinetoplast DNA1 is a unique extrachromosomal DNA network found in the single mitochondrion of parasitic flagellated protozoa of the family Trypanosomatidae
A Protein in C. fasciculata That Interacts with a Unique OriH-associated Sequence—It has been previously suggested that discontinuous replication of the minicircle H-strand initiates at or near the conserved hexameric sequence 5ЈACGCCC-3Ј, of the displaced parental L-strand [21, 22], at the proposed H-strand replication origin
A protein that interacts with this unique site in Kinetoplast DNA (kDNA) minicircles has been detected in 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. Protein Purification—Protein purification was carried out following the specific binding of the oriH-associated 14-mer sequence (H14, 5ЈGTAGGGGCGTTCTG-3Ј) oligonucleotide, by the mobility shift electrophoretic assay, as described below. One unit of UMSBP is defined as the amount of protein required for binding of 1 fmol of the oriL-associated UMS (5Ј-GGGGTTGGTGTA-3Ј) or the oriH-associated 14-mer (H14, 5Ј-GTAGGGGCGTTCTG-3Ј) DNA ligands, under the standard mobility shift assay conditions. Experiments were carried out under the standard mobility shift assay conditions, by serial dilutions of both UMSBP and the oligonucleotide probe, while keeping constant the molar ratio of added protein [Padded] to total DNA [DNAtotal] and varying the total concentration. The fraction () of protein that is active in DNA binding, as judged by the ability to form a protein-DNA complex band in gel electrophoresis, is calculated from ␣, the total concentration of DNA, and the concentration of added protein using the equation:  ؍ ␣[DNAtotal]/[Padded] [38]. DNA binding activity was assayed under the standard binding assay conditions as we have previously described [28,29], using the electrophoretic mobility shift analysis, and quantified using phosphorimaging, as described under “Experimental Procedures.” The 32P-labeled H14 5Ј-GTAGGGGCGTTCTG-3Ј and UMS 5Ј-GGGGTTGGTGTA-3Ј (10.6 fmol/assay) were used as radioactive ligands
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