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Title: Functional characterisation of an evolutionary conserved domain of non-coding Y RNA in human chromosomal DNA replication
Author: Gardiner, T. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2008
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In order to investigate the function of the Y RNAs, I employed a human cell-free DNA replication assay. Firstly, I used this assay to verify the original observations that showed Y RNAs to be required for human chromosomal DNA replication in vitro. I then carried out density substitution experiments to show that Y RNA mediated DNA replication in human nuclei was semi-conservative. I also used a mouse cell-free system to show that Y RNA function in DNA replication can act independently of the chemical synchronisation used to prepare human nuclei and to show that Y RNA function is conserved between mouse and man. The Y RNAs are highly conserved both in terms of sequence and structure in the vertebrates. The Y RNAs are also found in some non-vertebrate organisms such as C. elegans, B. floridae and D. radiodurans although they are not found in yeast or flies. I subsequently studied the evolutionary conservation of the Y RNAs further by using the cell free system to test the functionality of several different species’ Y RNAs. I concluded that Y RNA function is conserved throughout vertebrate evolution. RNA to promote DNA replication appeared to correlate with conserved sequence domains in the double stranded stem of the RNA. From this information I generated a library of mutant hY1 RNAs containing alterations to their sequence and structure to investigate further which domains were essential for DNA replication. Mutant Y RNA with deletions of the loop and secondary stems promoted DNA replication as efficiently as wild-type human Y1 RNA. Deletions in the upper-stem of the RNA, however, did not allow DNA replication to proceed. Further alterations to the sequence of the upper stem in most cases abrogated function in DNA replication. The upper part of this double-stranded stem of hY1 RNA was not only necessary, but also sufficient for Y RNA function in DNA replication. DNA oligonucleotides were not able to substitute for human Y RNA in vitro.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available