Final Report Summary - ORICODE (Unraveling the code of DNA replication origins and its link with cell identity)
Cells divide and transmit their genetic information (the DNA) through a process called DNA replication. This occurs during the development of a fertilized egg into an adult constituted of about 30 trillion cells and also all along adult life. DNA replication is an exquisitely regulated process that allows chromosomes to be copied from about 30 000 sites dispersed along the genome and called “DNA replication origins”. Errors in DNA duplication lead to genome instability or mutations that can cause abnormal development or genetic diseases. The characteristics of replication origins have remained poorly defined, preventing the full understanding of a number of diseases, including cancer. We wanted to unravel the replication origin code, to determine how this code may regulate cell fate or cell identity and to isolate new proteins involved in the recognition of this code.
To this aim, we developed a new procedure to map DNA replication origins genome-wide based on an improved experimental method to isolate the first pieces of DNA made at replication origins and on a robust bioinformatics analysis of these data. Our results have permitted to unravel to an unprecedented level the main genetic and epigenetic features of replication origins, some of which were unexpected.
1. Replication origins are in large excess along the chromosomes. This allows cells to choose which ones will be activated according to the cell type, cell growth conditions, cell fate, or in response to DNA damage.
2. Replication origins are enriched at chromosomal regions that are highly transcribed.
3. Replication origins have in common a new genetic element, called OGRE, for Origin G-rich Repeated Element. These elements form four-stranded structures called G quadruplexes. They are located 280 bp upstream of the initiation sites of DNA synthesis, and are exposed sites on the chromosomes. In addition to G-rich motifs, a CACA repeat motif was also identified.
4. DNA sequences with OGRE elements can be used to create new replication origins.
5. Specific chromatin features, including open chromosomal regions and developmental-specific chromosome marks (such as Polycomb marks), are tightly associated with replication origins.
6. Cells engaged in different differentiation programmes have specific subsets of replication origins. The pattern of replication origins is reprogrammed during dedifferentiation of mouse cells.
7. DNA replication origins appear to be localized at the interface of chromatin loops and high-salt and DNase-resistant chromatin structures. These chromatin structures are strongly associated with late replication domains and with lamin B1.
We have also employed several methods that make use of Xenopus laevis egg extracts or cell extracts and tagged proteins to identify new proteins involved in DNA replication. We discovered Obi1, a completely new factor that is involved in the regulation of the replication initiation complex and that could be implicated in a dwarfism syndrome. Finally, we unexpectedly found that MCM9 interacts with proteins involved in the repair of mismatched bases on DNA, and provided evidence that MCM9 could help eliminating the mismatched bases before DNA repair. Other new factors identified by our screens are under analysis.
To this aim, we developed a new procedure to map DNA replication origins genome-wide based on an improved experimental method to isolate the first pieces of DNA made at replication origins and on a robust bioinformatics analysis of these data. Our results have permitted to unravel to an unprecedented level the main genetic and epigenetic features of replication origins, some of which were unexpected.
1. Replication origins are in large excess along the chromosomes. This allows cells to choose which ones will be activated according to the cell type, cell growth conditions, cell fate, or in response to DNA damage.
2. Replication origins are enriched at chromosomal regions that are highly transcribed.
3. Replication origins have in common a new genetic element, called OGRE, for Origin G-rich Repeated Element. These elements form four-stranded structures called G quadruplexes. They are located 280 bp upstream of the initiation sites of DNA synthesis, and are exposed sites on the chromosomes. In addition to G-rich motifs, a CACA repeat motif was also identified.
4. DNA sequences with OGRE elements can be used to create new replication origins.
5. Specific chromatin features, including open chromosomal regions and developmental-specific chromosome marks (such as Polycomb marks), are tightly associated with replication origins.
6. Cells engaged in different differentiation programmes have specific subsets of replication origins. The pattern of replication origins is reprogrammed during dedifferentiation of mouse cells.
7. DNA replication origins appear to be localized at the interface of chromatin loops and high-salt and DNase-resistant chromatin structures. These chromatin structures are strongly associated with late replication domains and with lamin B1.
We have also employed several methods that make use of Xenopus laevis egg extracts or cell extracts and tagged proteins to identify new proteins involved in DNA replication. We discovered Obi1, a completely new factor that is involved in the regulation of the replication initiation complex and that could be implicated in a dwarfism syndrome. Finally, we unexpectedly found that MCM9 interacts with proteins involved in the repair of mismatched bases on DNA, and provided evidence that MCM9 could help eliminating the mismatched bases before DNA repair. Other new factors identified by our screens are under analysis.