Dr Lousie Boisen, Research Fellow, Biomedical Cell Biology Division at Warwick, will present this seminar, speaking on 'Genome-wide sequencing of Okazaki fragments in S. pombe'.

Abstract

DNA replication is a semi-discontinuous process, where the leading strand is synthesised continuously and the lagging strand is synthesised as a series of small fragments, known as Okazaki fragments.

Okazaki fragment synthesis is initiated when a primase synthesises a short RNA primer, which is then extended by DNA polymerases. In general, priming is assumed to be random. The exceptions are at replication origins on the leading strand and at replication fork barriers (RFB) on the lagging strand.

We are currently working on a project aimed at further investigating the nature of priming sites in the fission yeast genome. By doing a genome-wide sequencing of Okazaki fragments from wild-type yeast and strains with mutations in replication-associated genes we can determine whether priming is completely sequence independent or if any specificity exists. Furthermore, this analysis should allow us to identify replication fork barriers (RFB) in the genome.

The analysis is centred on the sequencing of Okazaki fragments isolated from logarithmically growing fission yeast strains. The DNA is purified using standard methods and the Okazaki fragments isolated through an alkaline sucrose gradient. To prepare the single-stranded Okazaki fragments for sequencing, we have developed a method for making them double-stranded without losing sequence information at either end. This will allow us to obtain as much information as possible from the sequencing.

This technique has the potential to become a valuable tool to identify RFB in different species. As the resolution of stalled replication forks can lead to genomic instability, this would be important for increasing our understanding of genomically based diseases such as cancer.

Further information

All staff and students are welcome and there is no need to register in advance. If you have any queries, please contact Gemma Wild at G dot Wild at warwick dot ac dot uk

This research seminar is part of the Biomedical Cell Biology Seminar series, presented by the Division of Biomedical Cell Biology, Warwick Medical School.