Principal Supervisor: Dr. Marco Saponaro, Institute of Cancer and Genomic Sciences
Co-supervisor: Dr. Martin Higgs, Institute of Cancer and Genomic Sciences
PhD project title: Crosstalk between RNA Pol II transcription and DNA replication
The DNA in our cells is a substrate for both RNA Pol II (RNAPII) transcription and DNA replication. Cells perform RNAPII transcription from the G1 to the G2 phases of the cell cycle and transcription of a gene is actually the most efficient mechanism to regulate the expression of its product. However, there is also a moment in the life of a cell that the DNA used by RNAPII becomes also the template for DNA replication, a compulsory step when cells decide to duplicate. Consequently, at any given time only one of the two processes can use the DNA as a substrate. We know that the two processes of transcription and replication are connected and that they need to be precisely coordinated to avoid reciprocal interference, but how this is actually achieved is unknown. We understand however that (i) the actual process of transcription can induce DNA damage, (ii) genome instability is greatly increased in contexts with defective transcription and (iii) in these contexts DNA replication is directly impaired leading to genome instability. This problem has enormous relevance, not only because it is a fundamental question at the heart of the cell biology, but also because these processes directly impact on human health, being associated with cancer development and neurological disorders. Over the years, a great deal of attention has been focused on identifying individual transcription-associated factors that mediate increased genome instability. However the global impact of normal RNAPII transcription on DNA replication has been largely ignored.
Aims of the project and methods:
We intend to answer at the systemic level how RNAPII transcription and DNA replication manage to coexist on the DNA template without interfering with each other, understanding how these processes are regulated in space and time. In this project we will address some fundamental questions in the field, principally: i) how transcription and replication are coordinated in order to avoid collisions; ii) how DNA replication adapts when encountering defective transcription; iii) whether transcription and replication are always in conflict, or whether there are instances in which they can support each other, and which mechanisms control it. We will also characterise how these processes crosstalk to influence each other, and how cells react in the event that RNAPII and the DNA replication machinery collide. In order to deliver these complex answers, we will make use of a combination of multi-disciplinary, cutting edge approaches. These will include:
- Genome-wide next generation sequencing analyses of both transcription and DNA replication, to determine their occupancies in space and time;
- Cell biology studies to characterise the impact of the interference between transcription and replication on genome stability (immune-fluorescence staining of DNA damage sites, pulse-field electrophoresis, activation of the DNA damage response by immunoblotting);
- Functional studies to characterise the roles of factors involved in coordinating, and resolving conflicting situations, between these two processes (impact of the RNAi of specific factors on genome-wide coordination between transcription and replication and on the DNA damage levels).
- Saponaro et al., RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress. Cell 2014; doi: 10.1016/j.cell.2014.03.048
- Kantidakis et al., Mutation of cancer driver MLL2 results in transcription stress and genome instability. Genes & Dev 2016; doi: 10.1101/gad.275453.115.
- Higgs et al., BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks. Mol Cell 2015; doi: 10.1016/j.molcel.2015.06.007.
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- Cell culture
- Genome wide next generation sequencing approaches like RNA-Seq, GRO-Seq, ChIP-Seq and Repli-Seq
- RNAi techniques
- Fluorescent and visual microscopy
- Pulse-field electrophoresis
- Immunoblotting and immunofluorescence
- Molecular cloning
Contact: Dr. Marco Saponaro, Institute of Cancer and Genomic Sciences