Jordan Sanders, Schwacha Lab
Investigating chromosomal common fragile site breaks in S. cerevisiae
DNA double strand breaks occur frequently in regions of chromosomes known as common fragile sites (CFS). Though breakage at some of these sites is present in a number of human cancers, the mechanism by which these breaks arise is poorly understood. Mutation to the primary sensor kinase of the S-phase replication checkpoint, ATR/Mec1, causes an increase in the incidence of these CFS breaks in both humans and S. cerevisiae. Most interestingly, CFS breaks will only arise in cells entering metaphase/anaphase, indicating that these breaks are not caused by simple replication defects in S-phase. CFS breaks also do not occur merely as a result of the separation of sister chromatids that are not fully replicated. As such, it remains to be ascertained as to what exactly causes these fragile site breaks, and how ATR/Mec1 prevents the accumulation of this particular type of DNA damage.
The Schwacha lab has isolated a mutation in one of the subunits of the Mcm replicative helicase, mcm2-DENQ, which offers an explanation of how these fragile site breaks arise. mcm2-DENQ S. cerevisiae exhibit DNA damage accumulation kinetics in a manner identical to that of mec1 mutant cells, suggesting that Mec1 and the Mcm complex work together to prevent CFS breaks. However, mcm2-DENQ strains to not accumulate visible CFS breaks as mec1 mutants do, probably due to the fact that mcm2-DENQ cells are able to repair breaks, while mec1 mutants are not.
In an effort to test the hypothesis that Mec1 and the Mcm complex work in concert to prevent CFS breaks, I have generated a number of strains that interfere with DNA repair mechanisms in order to try to isolate CFS breaks in mcm2-DENQ. In addition, we have been investigating the identity of the enzyme(s) responsible for causing CFS breaks in G2/M. Immunofluorescence detecting hallmarks of DNA damage, as well as pulsed field gel electrophoresis are used to assess various candidate strains for the indicators of CFS breaks.
Friday, December 7th
A219B Langley Hall