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L. Fabiani - DNA replication mechanisms and genome stability in Saccharomyces cerevisiae
level of the fork to give the correct conformation
through the cohesin ring. In replication stress condition
and in the presence of DNA damage Chl1p could
contribute to protect the fork from the formation of
ricombinogenic intermediates and to promote the
DNA replication restart.
During these first months of financial support we
identified the mutation present in ofm5 by the gap
repair technique to recover and finally sequence the
mutated copy of CHL1 gene in ofm5. The sequencing
of CHL1 in both ofm5 and wildtype strains
showed the presence of a point mutation in chl1 ofm5
allele, a transition of G to A at the level of
nucleotide 275 of the coding sequence, converting a
Trp codon (TGG) at the aminoacid 92 of the protein
to a stop codon (TAG). This premature stop codon
cause a null allele: all the important helicase domains
are lost. We obtained the chl1 deletion mutant and
analyzed its sensitivity to DNA-damaging agents.
chl1 is sensitive to hydroxyurea (HU) (depletion of
dNTP pools), methylmethanesulfonate (MMS) (alkylation
of bases) and UV radiation (pyrimidine dimer
formation). We analyzed then the correct activation
of the S phase checkpoints, the replication and intra-
S checkpoints, after synchronization in G2 with
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nocodazole and release, respectively, in the presence
of HU (S phase arrest in response to replication
blocks) and MMS (slowing of replication in response
to DNA damage in S phase). Both checkpoint mechanisms
are efficiently activated.
In the future, with the purpose to understand the
potential role of Chl1p at the replication fork, we will
analyze, by two-dimensional gel electrophoresis, the
replication intermediates in both wildtype and chl1
deletion mutant, at the level of chromosome III,
before and after treatment with DNA damaging
agents (HU, MMS), and at the level of the region of
chromosome XII containing the cluster of rDNA.
rDNA is a good model to study the different steps of
DNA replication and genome stability beeing one of
the most fragile sites of the genome. It will be then
interesting to verify the presence of Chl1p at the
level of the replication fork by ChIP (Chromatin
Immuno-Precitation) experiments by adding a tag
sequence at the C-terminus of the protein. The presence
of a tag will allow us to check the presence of
post-translational modifications (phosphorylation,
sumoylation, ubiquitinylation and acetylation) of
Chl1p under normal and in response to DNA damage
conditions.