We have achieved the objectives of the proposal by developing novel molecular biology and biochemistry approaches and by performing single-molecule experiments that have shed light in the dynamic aspects of the different phases of the eukaryotic replication reaction, namely the loading and the activation phases.
For example, we employed novel integrated force and fluorescence single-molecule experiments and reconstitution of purified proteins from S. cerevisiae to show that the loading intermediates are generally mobile, but that their mobility is reduced at the origin, leading to their retention there. Our results, showed that the proteins involved in the loading phase of DNA replication are mobile in character; that the origin, well known to be preferred by ORC, can reduce this mobility; and that this generally favors the build-up of MCM DH there. We also show that many MCM SH are loaded onto DNA, and that both they and the MCM DH are mobile. The large abundance of the MCM SH species suggests a lack of coordination in the loading of MCM hexamers, and may indicate a requirement for mechanisms that remove unproductive MCM SH. Follow-up studies related to these findings are in progress.
We have further repeated these experiments on DNA molecules that include an origin that can be chromatinized by reconstituting a single nucleosome on both of its flanks. These experiments indicated that a chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint. This contributes to our understanding of how DNA replication is influenced by the chromatin context, which is a fundamentally important and active area of investigation.
Additionally, in our study of the activation phase, we set out to perform in vitro reconstitution of the S. cerevisiae CMG helicase through its canonical pathway at the single-molecule level. Once this was achieved, we provided new insights on the link between CMG mobility and nucleotide binding. Our results indicated that nucleotide binding by CMG halts its diffusive motion on double-stranded (ds)DNA in a manner that does not involve DNA melting. Furthermore, our results suggested that cellular ATP prevents newly assembled CMG from diffusing on dsDNA. These findings from single-molecule and ensemble experiments are summarized in a model that can fully explain the nucleotide dependence of CMG helicase mobility.
The above-mentioned results have been published in journal articles and presented at scientific lectures either for specialists or for the general public. The project also led to several technical advances in biochemistry and biophysics, which have contributed to both collaborative and independent projects that make use of and/or describe these advances, which have likewise been published in journal articles.
