Binding of the Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

Itay Onn,Irit Kapeller,Kawther Abu-Elneel,Joseph Shlomai

doi:10.1074/jbc.m606374200

Itay Onn, Irit Kapeller + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m606374200

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Abstract

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.

Highlights

Kinetoplast DNA4 is a unique extrachromosomal DNA found in the single mitochondrion of trypanosomatids
UMS-binding protein (UMSBP) Interacts in Vivo with Kinetoplast DNA (kDNA) Networks—UMSBP was immunolocalized within the kinetoplast to two discrete foci located at the kineto-flagellar zone [24] at the site implicated with kDNA minicircle replication initiation [11]
The minor slower migrating bands observed may represent traces of higher oligomeric forms of UMSBP, generated under the cross-linking conditions, that resisted the denaturing and reducing electrophoresis conditions. These results indicate that UMSBP interacts in vivo with kDNA networks

Summary

EXPERIMENTAL PROCEDURES

Preparation of UMSBP—Cloning of the C. fasciculata UMSBP gene open reading frame [15] and the preparation of pure recombinant UMSBP were conducted as we have previously described [23]. In the second step the fragments described above were used as template with the primers set A (supplemental Table 2), yielding a 313-bp fragment that included a WT H14 and mutated UMS sequences (positions 489 –500). Micrococcal Nuclease Assay—Samples of UMSBP as indicated were added to a reaction mixture containing 0.25 pmol of 5Ј-32P-labeled 313-bp minicircle DNA fragment (positions 319 – 631, GenBankTM accession number M19266), 25 mM Tris-Cl, pH 7.5, 2 mM MgCl2, 5 mM CaCl2, and 20 mM dithiothreitol. Analysis of Proteins by SDS-PAGE—Protein samples in loading buffer containing 50 mM Tris-Cl, pH 6.85, 4% SDS, 3.5% ␤-mercaptoethanol, 10% (v/v) glycerol, and 10 mM EDTA were incubated at 37 °C for 30 min and loaded onto a 16.5% TrisTricine SDS-polyacrylamide gel [31] along with protein size markers (Rainbow pre-stained low molecular weight, Amersham Biosciences). Membranes were probed for 45 min with a 1:13,000 dilution horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (DacoCytonation) followed by ECL detection as recommended by the manufacturer (Amersham Biosciences)

RESULTS

Interactions of UMSBP with Its Two Binding Sites at the Minicircle

DISCUSSION