Final Report Summary - DNAMEREP (The role of essential DNA metabolism genes in vertebrate chromosome replication)
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Stand alone description of the project and its outcomes.
Over the years my group has successfully identified the role of key vertebrate essential DNA metabolism factors involved in DNA replication and DNA repair. To this end we set up novel assays to elucidate the molecular role of essential DNA repair and DNA replication proteins. In particular we focused on the roles of these factors at chromosome loci that require the special assistance of DNA repair genes to be replicated. Using this approach, we reconstituted for the first time the replication, the protein composition and the structure of the chromatin associated to human centromeres. We then identified and isolated the Xenopus ortholog of BRCA2 tumor suppressor gene and demonstrated that in the absence of BRCA2 DNA replication forks accumulate abnormal levels of single stranded DNA. We revealed the molecular mechanisms by which BRCA2 and BRCA1 prevent extensive degradation of DNA and confirmed that this phenomenon occurs in human cancer cells. To this end we showed that SMARCAL1 helicase recognizes these gaps and promotes the formation of reversed forks, when these stall. Reverse forks constitute the entry point for Mre11 nuclease to initiate extensive nascent DNA degradation in the absence of BRCA2 and or BRCA1. We also showed that BRCA2 promotes the formation of stable RAD51 nucleofilaments that physically protect DNA from nuclease mediated degradation. This work highlighted the essential role of BRCA2 tumor suppressor during unchallenged DNA replication for the first time. We then revealed how replication origin formation is regulated in vertebrate cells by identifying the essential role of the FACT complex subunit SSRP1 in regulating the assembly of ORC complex onto DNA. We also showed that increased SSRP1 levels delay a key developmental stage known as mid-blastula transition (MBT), when fast embryonic cycles terminate and slower somatic cycles begin, and strikingly, accelerate post-MBT development by reducing the length of the cell cycle. These results will have significant impact on our understanding of the function of essential DNA metabolism genes as originally proposed.
Stand alone description of the project and its outcomes.
Over the years my group has successfully identified the role of key vertebrate essential DNA metabolism factors involved in DNA replication and DNA repair. To this end we set up novel assays to elucidate the molecular role of essential DNA repair and DNA replication proteins. In particular we focused on the roles of these factors at chromosome loci that require the special assistance of DNA repair genes to be replicated. Using this approach, we reconstituted for the first time the replication, the protein composition and the structure of the chromatin associated to human centromeres. We then identified and isolated the Xenopus ortholog of BRCA2 tumor suppressor gene and demonstrated that in the absence of BRCA2 DNA replication forks accumulate abnormal levels of single stranded DNA. We revealed the molecular mechanisms by which BRCA2 and BRCA1 prevent extensive degradation of DNA and confirmed that this phenomenon occurs in human cancer cells. To this end we showed that SMARCAL1 helicase recognizes these gaps and promotes the formation of reversed forks, when these stall. Reverse forks constitute the entry point for Mre11 nuclease to initiate extensive nascent DNA degradation in the absence of BRCA2 and or BRCA1. We also showed that BRCA2 promotes the formation of stable RAD51 nucleofilaments that physically protect DNA from nuclease mediated degradation. This work highlighted the essential role of BRCA2 tumor suppressor during unchallenged DNA replication for the first time. We then revealed how replication origin formation is regulated in vertebrate cells by identifying the essential role of the FACT complex subunit SSRP1 in regulating the assembly of ORC complex onto DNA. We also showed that increased SSRP1 levels delay a key developmental stage known as mid-blastula transition (MBT), when fast embryonic cycles terminate and slower somatic cycles begin, and strikingly, accelerate post-MBT development by reducing the length of the cell cycle. These results will have significant impact on our understanding of the function of essential DNA metabolism genes as originally proposed.