Title: Telomere Replication and Stability
Funder: Medical Research Council

Abstract: The formation and/or metabolism of DNA secondary structures is important for many physiological processes, and is particularly relevant during DNA replication, transcription and repair. However, persistent or aberrantly processed DNA secondary structures can have pathological consequences and are an established source of genome instability. DNA secondary structures can form from alternative DNA sequence motifs (eg. trinucleotide repeats or guanine rich DNA that form four stranded DNA structures called G-quadruplexes (G4)) or as an intermediate formed during DNA replication, repair or recombination. Bioinformatics’ data has demonstrated that DNA secondary structures motifs are conserved throughout evolution. Furthermore, the discovery of specialised factors required for dismantling or bypassing such DNA structures, together with the direct observation of G4 focus formation in human cells strongly supports their existence in vivo. Efficient genome-wide replication requires not only the replicative polymerase but also bypass polymerases, dismantling helicases and repair activities, which engage with the replisome when it encounters DNA damage, topological constraints, transcription complexes and DNA secondary structures. Failure to stabilize, repair or restart the replication fork is a potential source of genome instability, the hallmark of many diseases including cancer. Thus, the cell possesses mechanisms to regulate both the disassembly of DNA secondary structures during DNA replication and the re-establishment of the DNA or chromatin marks after duplication. Evidence is emerging that several replication and repair proteins impact on telomere replication to avert telomere fragility, but the nature, cause and links to human disease remain unknown. Ultimately, the program of research comprises complementary multidisciplinary approaches that will encompass molecular and cellular biology, proteomics and vertebrate genetics (mouse and human cells) to discover and examine new genes required for faithful replication at telomeres and throughout the genome. The research will provide a framework for comprehending the contributions of replication factors in general DNA replication and cancer in humans. First, I propose to investigate the enzymatic activities of known and new factors that suppress replication stress at telomeres. Telomere replication stress displays as multiple spatially distinct telomere foci induced by replication stress (aphidicolin) along common fragile sites (CFS) present as chromosome discontinuities. This raises the possibility that telomere-linked replication problems are processed similarly to CFS. Stable shRNA knock-down or generated Knock-Out mammalian cell lines will help evaluate the genetic interactions of the known nucleases MUS81 and XPF in the suppression of telomere fragility similarly or not to the suppression of CFS. In order to identify new factors responsible for the induction of telomere fragility in response to replication stress, I will employ proteomics of isolated chromatin (PICh) to identify the factors that are recruited and/or excluded specifically to fragile telomeres. This will be carried out in collaboration with Peter Faull, head of Biological Mass Spectrometry and Proteomics Laboratory at MRC-CSC. Candidates will be then confirmed and characterised by shRNA knock-down to study their role in maintaining genome stability and telomere homeostasis.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS