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Title: Mitochondrial DNA replication in the sea urchin Strongylocentrotus purpuratus
Author: Mayhook, Andrew Geoffrey
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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In animal development, the normal regulation of mitochondrial DNA (mtDNA) replication, which ensures a doubling of the amount of mtDNA per cell division, is altered. In oogenesis there is extensive mtDNA synthesis, but no nuclear DNA replication, whereas the situation is reversed in embryogenesis. The aim of this project was to provide basic information about the molecular mechanism of mtDNA replication in the sea urchin Strongylocentrotus purpuratus, so that the question of what regulates mtDNA replication can then be addressed. The two-dimensional (2D) agarose gel electrophoresis system developed by Brewer and Fangman (1987) was used to analyse the structures of the replicating mtDNA molecules. This gel system exploits the fact that non-linear DNA molecules are retarded under gel electrophoresis compared to linear DNA molecules. The DNA moleules are subjected to restriction enzyme digestion before electrophoresis, and curves of different, characteristic shapes are generated by replication intermediates (RIs) of different structures. Different restriction enzyme digestions allow the pattern of replication fork movement to be related to the positions of the restriction sites within the genome, which enables the position of the replication origin to be located in the DNA molecules. The gel electrophoretic analyses provided a complex, but internally consistent set of results, giving a picture of the replication process in S. purpuratus mtDNA molecules. The analyses revealed that the replication origin for leading-strand synthesis was located within the non-coding region of the genome, and that replication was unidirectional, by a strand-displacement mechanism, and occurred towards the 12S rRNA gene. The 2D gel experiments also revealed that pause sites for leading-strand synthesis were located at several sites in the mitochondrial genome. The most prominent of these pause sites occurred close to a prominent lagging-strand replication origin, in the region of the genome near the boundary between the genes for subunit 6 of ATP synthase (A6), and subunit HI of cytochrome c oxidase (COIII). RNase protection experiments, performed using probes covering the noncoding region of the mtDNA, mapped the 5' end of the nascent DNA strands to nt 1150 +/-10, and suggested that the 3' end of lagging-strand molecules also mapped to the same location, implying a pause site for lagging-strand synthesis at the leading-strand origin. The ends of the DNA molecules corresponding to the leading-strand replication pause site, and the origin of lagging-strand synthesis in the A6/COIII region of the genome were, however, below the level of detection of the RNase protection experiments. The ends of the transcripts for the A6 and COIII genes were mapped, which revealed one major and one minor 3' end for the A6 RNA, and a single discrete 5' end for the COIII RNA. From the sizes of the molecules identified by the RNase protection experiments, it appeared that the ends of the 2 transcripts were very close together. An in vitro transcription system was used to investigate whether there is a link between transcriptional control and DNA replication in sea urchin mitochondria. A possible link between the 2 processes was suggested by the location of some of the replication pause sites close to the 3' ends of genes: sites where transcription termination could be involved in the production of transcripts. The in vitro transcription system used bacteriophage RNA polymerase, because the enzyme had not been isolated from S. purpuratus mitochondria at the time the experiments were performed. Bacteriophages have single subunit RNA polymerases, which show considerable sequence similarity at the amino acid (aa) level to the mtRNA polymerase isolated from yeast (Masters et al, 1987). Transcription run-off assays were carried out using the region of the mtDNA containing the A6/COIII gene boundary, because this portion of the genome contains both a replication pause site and a lagging-strand replication origin, as well as being a possible site for transcription termination. When transcription of the mtDNA was carried out in the same direction as RNA synthesis in vivo, 6 sites for transcription termination were detected. However, no sites were detected when transcription through the mtDNA occurred in the opposite direction. The termination sites did not correspond with the 3' end of the A6 transcript detected in vivo by the RNase protection experiments. This would, therefore, imply that if these sites were acting to terminate transcription in vivo, then further processing of the transcript would have to occur to generate the mature mRNA. Although 2 DNA-binding proteins which act close to the A6/COIII gene boundary have been identified in mitochondrial protein extracts (S.A. Qureshi & H.T. Jacobs, unpublished data), the addition of these protein extracts (prepared by SAQ) to the in vitro transcription reactions did not appear to affect the pattern of transcription termination. On the basis of the gel electrophoretic data, I propose a model for mtDNA replication in S. purpuratus (Mayhook et al, 1992). The origin of leading-strand replication is located in the non-coding region of the genome at nt 1150 + 10. DNA synthesis is initially unidirectional, by strand displacement (as proposed for vertebrate mtDNA; Clayton, 1982), towards the 12S rRNA gene. The gel electrophoretic data are consistent with the electron microscopy data of Matsumoto et al (1974), which imply that multiple origins exist for lagging-strand synthesis. A prominent lagging-strand origin was detected near the A6/COIII gene boundary, and its occurrence in the same region of the genome as a pause site for leading-strand synthesis suggests that the pausing of leading-strand replication may have a role in the initiation of lagging-strand synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available