Current affiliations

• Position

  Post Doc

  Company

  Cancer Research UK

  Further information

City:

None chosen

Hub:

London

Anti-cancer research, Drug discovery, oncology, DNA damage and repair, cell cycle checkpoints

In budding yeast, the S-phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Under conditions of limiting dNTP levels, replication fork stalls and continued unwinding of DNA results in the formation of RPA-coated single-stranded (ss) DNA, which contribute toward activation of Mec1 and Rad53 kinases to carry out S-phase responses. Mrc1 has been shown to play an important role in both fork stalling and checkpoint activation. Given that continued unwinding of DNA and the general belief in requirement for RPA coated ssDNA to be a prerequisite for checkpoint activation, we were particularly interested in deciphering the outcome of preventing unwinding or losing RPA during S phase. We made use of heat-inducible degron mutants to assess the consequences of losing the presumptive replicative helicase during S phase which should cause fork stalling while limiting the generation of ssDNA. Techniques used included:
• Targeted gene disruption by PCR and yeast transformation
• Generation of new yeast strains by mating and tetrad dissection
• Radioactive in situ kinase assay
• Fluoroscence associated cell cytometry
• Two-dimensional agarose gel electrophoresis
• Southern blot analysis of replication intermediates