Principal Supervisor: Dr Marco SaponaroLink opens in a new window

Co-supervisor: Dr Martin Higgs

PhD project title: G2/M DNA synthesis and its implications for transcription regulation and genome stability

University of Registration: University of Birmingham

Project outline:

RNA Pol II transcription and DNA replication are the two essential processes that use the DNA in our cells as a substrate, allowing them to express the content of their genetic information and to propagate these instructions to daughter cells. However, the DNA can be engaged only by one of these processes at any given time, and we know that transcription can impair DNA replication inducing DNA damage and genome instability. Indeed, we recently discovered that also during the replication of genes transcription is still active, and consequently these regions are skipped and not replicated efficiently. Often, once a region is skipped it remains under-replicated throughout the cell cycle, becoming definitely duplicated only once cells are preparing for mitosis, in G2/M. This G2/M DNA synthesis (G-MiDS) is highly frequent in cells but not associated with sites of DNA damage nor regulated by canonical DNA damage repair factors, making it a totally new process. Importantly, it is an essential process for cells, as interfering with G-MiDS leads to an increase of mitotic problems, and G-MiDS sites overlap with hotspots of rearrangements identified in cancer cells. Altogether, we intend understanding what G-MiDS is, what factors are involved in this process, and the consequences for transcription and genome instability if G-MiDS is affected.

Aims of the project and methods:

For all these reasons, we are interested in investigating into more details what factors regulate and perform G-MiDS, and the consequences for cells from defective G-MiDS.

1. Identification and characterisation of factors and processes involved in G-MiDS, both through functional studies and by identifying proteins present at sites of DNA synthesis;
2. Cell biology studies to characterise the impact of the deregulation of factors involved in G-MiDS on genome stability (immune-fluorescence staining of DNA damage sites, activation of the DNA damage response by immunoblotting);

• Molecular studies to characterise the impact of G-MiDS and its deregulation on the expression of G-MIDS hotspot genes and their chromatin organisation (genome wide studies with bioinformatic analysis).

References:

Wang et al., Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M. Cell Reports 2021 Feb 16; 34(7): 108759.

Wang et al., Protocol for analysis of G2/M DNA synthesis in human cells. STAR Protocols 2021 May 29; 2(2): 100570.

Rojas et al., Positive and Negative Impact of RNA Pol II Transcription on DNA Replication Progression. SSRN

Williamson et al., UV irradiation induces a non-coding RNA that functionally opposes the protein encoded by the same gene. Cell 2017 Feb 23; 168(5): 843-855.e13.

Saponaro et al., RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress. Cell 2014 May 22; 157(5): 1037-49.

Bayley et al., H3K4 methylation by SETD1A/BOD1L facilitates RIF1-dependent NHEJ. Mol Cell 2022 May 19; 82(10): 1924-1939.e10.

Higgs et al., Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2. Mol Cell 2018 Jul 5; 71(1): 25-41.e6.

 

BBSRC Strategic Research Priority: Integrated Understanding of Health - Ageing

Techniques that will be undertaken during the project:

The student will have the opportunity to acquire a multiplicity of techniques including: cell culture; genome wide next generation sequencing approaches like RNA-Seq, and ChIP-Seq, with associated bioinformatic analysis; RNAi techniques; fluorescent and visual microscopy; immunoblotting and immunofluorescence, molecular cloning.

Contact: Dr Marco SaponaroLink opens in a new window