In all organisms the DNA content is precisely and efficiently replicated. DNA
replication starts at specific regions called replication origins, where a preReplicative Complex (pre-RC) is assembled at the end of the G2/M phase, maldng
chromatin competent for replication. At the transition between G1 and S phases the
activation of two conserved protein kinases triggers the transition from pre-RC into
the active form, the Replicative Complex (RC). Replication origins are not fired at
the same time during S phase, thus we can define origins as “early” and “late”
according to their time of activation. During the past years our knowledge about
DNA replication has improved due to the identification of novel proteins. Despite a
large body of work, it remains possible that additional factors remain unidentified.
The discovery of new proteins by genetic means is difficult due to the presence of
redundant mechanisms and to their requirement for cell survival. Thus the aim of my
project is to develop an unbiased assay to study, in Saccharomyces cerevisiae, the
proteome of a single eukaryotic replication origin and identify novel proteins
involved in DNA replication. Because the chromosomal environment profoundly
influences the behaviour of replication origins (such as timing, efficiency), it is
therefore of interest to analyse the proteomics of replication origins in their
endogenous context. Taking advantage of the pSRl recombination system, I
constructed two plasmids (pLTl/RsiteA and pLT2/RsiteB) that led to the integration
of recombination sites into the yeast genome and the excision in vivo of the late
replication origin ARS1413. These plasmids, allowing the excision of any flanking
region, represent important genetic tools. The resulting episome has been isolated
from the genome by density gradient purification in order to analyze by mass
spectrometry the protein associated with the specific replication origin. This
technique can be used to study the proteins involved in different steps of DNA
replication. In fact cells arrested in G1 with a-factor will provide the proteome of the
pre-RC while cells treated with HU will represent the proteome of late origins that
are inhibited by the S-phase checkpoint
