Within the project, we studied the restart of the replication fork (RF) at several barriers represented by a protein barrier and difficult to replicate G-quadruplex secondary DNA structures (G4s). For this purpose, we decided to use two complementary approaches: (1) Polymerase usage assay (Pu-seq) developed in our team, which allows us to follow DNA replication in vivo and (2) Chromatin Immunoprecipitation (ChIP) which detects recruitment of proteins to sites of interests.
Exploiting the techniques mentioned above, we have achieved the following results:
1) The newly formed RF travels over long region which allows us to study restarted RF. Around 50% of the restarted RF replicates 12 kb where they terminate with convergent canonical RF.
2) Restarted RF is more efficient in the removal of protein barriers than canonical RF.
3) When the RF stalls, the lagging strand is resected in order to generate a 3’-end for homologous recombination dependent restart. The resection is increased in a strain that has lost the DNA repair protein Ku70 (pKu70-). However, the leading strand is not degraded in pKu70- as well as in the wild type (pKu70+).
4) The restarted RF does not use the primase activity of Pol alpha.
5) We showed that interaction of PCNA with Cdc27 is not important for DNA polymerase (Pol) usage after the restart at RTS1, but instead may be important for its fidelity.
6) We observed substantial accumulation of DNA repair proteins Rad52 and Rpa3 close after the restart and a gradual decrease of both Rad52 and Rpa3
7) We demonstrated that accumulation of DNA polymerase Pol epsilon and the fork protection complex. Both accumulated before the restart site.
8) We explored the RTS1 model barrier and other DNA barriers as well. Initially, we wanted to use the ribosomal replication barrier to slow down the convergent replication fork. This would allow us to preserve the restarted RF for a longer period of time as the convergent canonical fork will be delayed. Nevertheless, we were surprised how inefficient rDNA barriers are in according to our Pu-seq analysis visualising DNA replication in millions of cells.
9) We studied the potential of G-quadruplex (G4) to stall DNA replication forks. Again, the effect on overall replication was negligible but in mutational assays we clearly see a tenfold increase when G4 is present.
10) In the end, I would like to stress that we established a collaboration with a French team at CRCM in Marseille, France, to study the effect of the telomeric CST complex on DNA replication genome-wide. We can clearly show that the CST complex affects Rif1 binding sites, Taz1-dependent heterochromatin islands and is involved in DNA replication of G4-containing DNA.
The above-mentioned results reached within this project were exploited and communicated during conferences and my public engagement activities. I shared them with other researchers as well as with students and colleagues/collaborators at the University of Sussex and worldwide at conferences and meetings.
Poster presentations at the following conferences conferences:
May 2017 - Replication, Recombination, Repair in Giens (France)
September 2017 - Eukaryotic DNA Replication and Genome Maintenance in Cold Spring Harbour (USA)
March 2018 – Marie Curie Alumini Association, meeting/conference in London (UK)
February 2019 – Mammalian DNA Repair in Ventura (USA)
May 2019 - Replication, Recombination, Repair in Giens (France)
Public engagement activities:
Since 2017 – Twitter account @Karel70951849
November 2017 – MEP pairing scheme organised by European Parliament
February 2018 – Brighton Science Festival
August 2018 – public outreach at Brighton Pride Festival
We have published a book chapter about Pu-seq method used in this study. (It does't contain data reached within this project and therefore it is not a direct output of this project)
Analysis of Replicative Polymerase Usage by Ribonucleotide Incorporation.
Keszthelyi A, Miyabe I, Ptasińska K, Daigaku Y, Naiman K, Carr AM.
Methods Mol Biol. 2018;1672:239-259.
We are currently preparing 3 manuscripts for publication containing the results of (1) restart of the DNA replication fork at the RTS1 barrier (2) the interaction between PCNA and cdc27 and (3) and Stn1 function in DNA replication (results described above). I will utilise the University of Sussex Research Archive (Sussex Research online –
http://sro.sussex.ac.uk/(si apre in una nuova finestra)) an online digital repository containing publically, freely available outputs from the staff at the University of Sussex. This database is indexed and used by Google Scholar or OpenDOAR. This will give access to our findings to a wide audience and therefore contribute to advertising the European Commission as a major funder for research and development.
All these actions will access and have accessed the results to wide audience and therefore contribute to advertising the European Commission as a major funder for research and development.
