Final Activity Report Summary - DNA REPAIR ARCHAEA (Analysis of the Initiation of DNA Recombination/Repair in thermophilic Archaea)
The ubiquitous Rad50 and Mre11 proteins were shown to be key actors of many processes involved in the maintenance of genome integrity in bacteria and eucarya, but their roles in the archaea, is presently unknown. My laboratory had previously showed that in most hyperthermophilic archaea rad50-mre11 genes were linked to nurA, encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and to herA, encoding a bipolar deoxyribonucleic acid (DNA) helicase suggesting the involvement of the four proteins in commune molecular pathways.
Since hyperthermophilic archaeal genetic tools are just emerging, I utilised immuno-detection approaches to get first in vivo data on the roles of these proteins in the hyperthermophilic species sulfolobus acidocaldarius. I firstly showed that the S. acidocaldarius strain was able to repair DNA fragmentation induced by high doses of gamma rays and I performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA, along with the RadA recombinase in both control and irradiated cells. I found that all proteins were synthesised constitutively, however each one displayed a specific sub-cellular partitioning.
Following gamma irradiation, I showed that both Mre11 and RadA were immediately recruited to DNA and were still DNA-bound in the course of DNA repair, whereas total levels of the HerA bipolar helicase were slightly increased. Furthermore, I showed, via immuno-precipitation assays, that Rad50, Mre11 and the HerA helicase interacted altogether in a constitutive way. These analyses demonstrated that, in S. acidocaldarius, the Mre11 protein was involved in post-irradiation DNA repair together with RadA recombinase and that this protein could play an active role in DNA repair processes and potentially act as a DNA damage sensor. Moreover, they demonstrated the functional interaction between Mre11, Rad50 and the HerA helicase and suggested that each protein played different roles when acting by its own or in association with partners.
This final activity report provided the first evidence for the implication of the Mre11 protein in DNA repair processes in archaea and for its interaction with both Rad50 and the HerA bipolar helicase. Further studies of the functional interactions between these proteins, the NurA nuclease and the RadA recombinase could allow for defining precisely the mechanism of action and the roles of these proteins in the archaea.
Since hyperthermophilic archaeal genetic tools are just emerging, I utilised immuno-detection approaches to get first in vivo data on the roles of these proteins in the hyperthermophilic species sulfolobus acidocaldarius. I firstly showed that the S. acidocaldarius strain was able to repair DNA fragmentation induced by high doses of gamma rays and I performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA, along with the RadA recombinase in both control and irradiated cells. I found that all proteins were synthesised constitutively, however each one displayed a specific sub-cellular partitioning.
Following gamma irradiation, I showed that both Mre11 and RadA were immediately recruited to DNA and were still DNA-bound in the course of DNA repair, whereas total levels of the HerA bipolar helicase were slightly increased. Furthermore, I showed, via immuno-precipitation assays, that Rad50, Mre11 and the HerA helicase interacted altogether in a constitutive way. These analyses demonstrated that, in S. acidocaldarius, the Mre11 protein was involved in post-irradiation DNA repair together with RadA recombinase and that this protein could play an active role in DNA repair processes and potentially act as a DNA damage sensor. Moreover, they demonstrated the functional interaction between Mre11, Rad50 and the HerA helicase and suggested that each protein played different roles when acting by its own or in association with partners.
This final activity report provided the first evidence for the implication of the Mre11 protein in DNA repair processes in archaea and for its interaction with both Rad50 and the HerA bipolar helicase. Further studies of the functional interactions between these proteins, the NurA nuclease and the RadA recombinase could allow for defining precisely the mechanism of action and the roles of these proteins in the archaea.