Bidirectional Replication Initiates at Sites Throughout the Mitochondrial Genome of Birds

Aurelio Reyes,Ming Yao Yang,Mark Bowmaker,Ian J Holt

doi:10.1074/jbc.m411916200

Aurelio Reyes, Ming Yao Yang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m411916200

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Abstract

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5' ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates.

Highlights

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA
The results indicate that bidirectional replication occurs at sites dispersed throughout the mitochondrial genome of G. gallus giving rise to replication forks that travel in both directions around the circle of mitochondrial DNA (mtDNA)
Origin Detection and Mapping—In a previous study, we reported that initiation of mtDNA replication encompasses a broad zone downstream of the 3Ј end of the D-loop, in mammals [10]

Summary

EXPERIMENTAL PROCEDURES

Purification of Chicken Liver Mitochondria—Chicken liver mitochondria were isolated first by a crude differential centrifugation procedure and subsequently on a single step sucrose density gradient. 20 –30 chick livers were minced finely with scissors and washed three or more times with 100 ml of homogenization buffer (HB) comprising 225 mM mannitol, 75 mM sucrose, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and 0.1% bovine serum albumin (fatty acid-free). Where indicated RNase H (Promega) treatment was 1 unit of enzyme for 1 h at 37 °C with 0.1–1.2 ␮g of mtDNA. RNase One (Promega) treatment was 5 units of enzyme for 10 min at 37 °C with 0.1–1.2 ␮g of mtDNA. In-gel digestion for fork-direction gels was carried out as follows, after separation of DNA in the first dimension, the gel lane was excised and washed twice with 10 mM Tris, 0.1 mM EDTA, pH 8.0, for 30 min at room temperature. Immobilized fragments of mouse mtDNA were detected by radiolabeled probes amplified via PCR, using the following pairs of primers: 5Ј-CAAAGGTTTGGTCCTGGCCT-3Ј and 5Ј-TGTAGCCCATTTCTTCCCA-3Ј np 69 –790; 5Ј-CACCTTCGAATTTGCAATTCG-3Ј and 5Ј-CTGTTCATCCTGTTCCTGCT-3Ј np 5,215–5,709; 5Ј-CGCCTAATCAACAACCGTCT-3Ј and 5Ј-TGGTAGCTGTTGGTGGGCTA-3Ј np 8,032– 8,497; and 5Ј-AACTGAACGCCTAAACGCAGGGA-3Ј and 5Ј-AACTGGATTTGAAGTTGCTAGGCA-3Ј np 13,867–14,518. Primers corresponding to G. gallus mtDNA were based on the published sequence [18]

RESULTS

Avian Mitochondrial DNA Replication

DISCUSSION