Final Report Summary - OXIDNA-REPAIR-CHROMA (Unraveling the role of Nucleotide Excision Repair factors and the dynamic of chromatin structure in the repair of oxidative DNA damages in vivo)
Our project was dedicated to a key and fundamental question related to one of the main medical challenge of the XXIst century in the European Union (EU) and western societies: ageing. The ageing population has considerably increased the incidence of cancer and neurodegnerations. These two major causes of mortality have been linked with oxidation of DNA bases by reactive oxygen species (ROS) (David et al., 2007).
DNA, in contrast to other component of cells like proteins or lipids are not renewable by de novo synthesis (Hoeijmakers, 2001). This unique property renders this macromolecule extremely sensitive to alterations and certainly explains why modifications of DNA can be so deleterious compared to oxidation of other macromolecules present in cells. The DNA in eukaryotic cells is packaged into chromatin to satisfy the space restriction in the nucleus but more importantly to organise the genome (Misteli and Soutoglou, 2009). This complex organisation of the DNA has a major influence on all DNA associated processes essential for the proper function of cells like transcription, replication and repair.
DNA repair mechanisms of oxidative base damages have been analysed extensively in vitro by using model DNA substrates and purified proteins or cell extracts. However, they did not reproduce the natural folding of the DNA in vivo. The processes that actually operate in response to oxidative base damages within cells remain largely unknown due to the complexity of generating base damage directly in vivo.
Base excision repair (BER) is the main repair pathway to eliminate abundant oxidative lesions such as 8-oxo-7,8-dihydroguanine (8-oxoG). However, there are several indications that a crosstalk between BER and at least several NER factors may be important in the protection of our genome against ROS. Mouse mutants for nucleotide excision repair (NER) revealed an unexpected link between DNA damages and premature aging. Interestingly, the premature aging phenotype is present without any additional external source of DNA damages, so it may be a consequence of DNA lesions caused by endogenous sources. The molecular mechanisms of this BER-NER crosstalk remain unknown and the existence of BER-NER crosstalk is even disputed by certain conflicting results.
Our goal was to molecularly dissect the involvement of NER proteins, in the repair of oxidative DNA damages in vivo.
Mechanisms of NER in living mammalian cells and the dynamic engagements of NER factors in DNA repair has been explored in our group by using live cell confocal microscopy, time-lapse imaging, photobleaching, and local DNA damage infliction (with multiphoton or UV-C laser). We have implemented these tools with a novel laser micro-irradiation technique generating oxidative DNA lesions (in the presence of a specific photosensitiser) and monitored the recruitment of NER-BER factors to such lesions.
This novel development, to generate oxidative DNA base damage, makes our study unique and allowed a very precise analysis in subnuclear structures of the nucleus. We particularly used this technique to generate oxidative DNA damage in the nucleoplasm and in the nucleolus to analyse the possible involvment of two NER initiating factors (CSB and XPC) in oxidative DNA damage repair.
We could clearly establish functionnal recruitment of these factors to our local oxidative DNA damage. The absence of detectable accumulation of downstream NER factors at the local oxidative DNA damage provide the first in vivo indication of the involvement of CSB and XPC in the repair of oxidative DNA lesions independent of the remainder of NER. Interestingly, CSB exhibited different and transcription-dependent kinetics in the two compartments studied (nucleolus and nucleoplasm), suggesting a direct transcription-dependent involvement of CSB in the repair of oxidative lesions associated with different RNA polymerases, but not involving other NER proteins.
Also the recruitment of BER factors like XRCC1 and OGG1 have been investigated and has lead to novel findings. The recruitment of OGG1 to 8-oxoG is not dependant on the presence of CSB; however, in absence of CSB and PARP activity no XRCC1 can be recruited to 8-oxoG. XRCC1 is a very important scaffold protein to allow efficient BER in vivo. The importance of XRCC1 for life is illustrated by the very early embryonic lethality of mice deficient for XRCC1 (Tebbs et al., 1999).
Our results led to a better understanding of the molecular mechanisms of oxidative base damage repair in vivo. The endogenously generated ROS can damage DNA in every organ and probably, more intensively in tissue with high oxygen consumption, like the brain. Our novel findings showing the possible redundancy of PARP and CSB for the efficient loading of XRCC1 may have a major impact to better counteract the deleterious effect of ageing, and oxidative DNA damage on the brain function in the elderly.
David, S.S. V.L. O'Shea, and S. Kundu. 2007. Base-excision repair of oxidative DNA damage. Nature. 447:941 - 50.
Hoeijmakers, J.H. 2001. Genome maintenance mechanisms for preventing cancer. Nature. 411:366 - 74.
Misteli, T., and E. Soutoglou. 2009. The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol. 10:243 - 54.
Tebbs, R.S. M.L. Flannery, J.J. Meneses, A. Hartmann, J.D. Tucker, L.H. Thompson, J.E. Cleaver, and R.A. Pedersen. 1999. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev Biol. 208:513 - 29
DNA, in contrast to other component of cells like proteins or lipids are not renewable by de novo synthesis (Hoeijmakers, 2001). This unique property renders this macromolecule extremely sensitive to alterations and certainly explains why modifications of DNA can be so deleterious compared to oxidation of other macromolecules present in cells. The DNA in eukaryotic cells is packaged into chromatin to satisfy the space restriction in the nucleus but more importantly to organise the genome (Misteli and Soutoglou, 2009). This complex organisation of the DNA has a major influence on all DNA associated processes essential for the proper function of cells like transcription, replication and repair.
DNA repair mechanisms of oxidative base damages have been analysed extensively in vitro by using model DNA substrates and purified proteins or cell extracts. However, they did not reproduce the natural folding of the DNA in vivo. The processes that actually operate in response to oxidative base damages within cells remain largely unknown due to the complexity of generating base damage directly in vivo.
Base excision repair (BER) is the main repair pathway to eliminate abundant oxidative lesions such as 8-oxo-7,8-dihydroguanine (8-oxoG). However, there are several indications that a crosstalk between BER and at least several NER factors may be important in the protection of our genome against ROS. Mouse mutants for nucleotide excision repair (NER) revealed an unexpected link between DNA damages and premature aging. Interestingly, the premature aging phenotype is present without any additional external source of DNA damages, so it may be a consequence of DNA lesions caused by endogenous sources. The molecular mechanisms of this BER-NER crosstalk remain unknown and the existence of BER-NER crosstalk is even disputed by certain conflicting results.
Our goal was to molecularly dissect the involvement of NER proteins, in the repair of oxidative DNA damages in vivo.
Mechanisms of NER in living mammalian cells and the dynamic engagements of NER factors in DNA repair has been explored in our group by using live cell confocal microscopy, time-lapse imaging, photobleaching, and local DNA damage infliction (with multiphoton or UV-C laser). We have implemented these tools with a novel laser micro-irradiation technique generating oxidative DNA lesions (in the presence of a specific photosensitiser) and monitored the recruitment of NER-BER factors to such lesions.
This novel development, to generate oxidative DNA base damage, makes our study unique and allowed a very precise analysis in subnuclear structures of the nucleus. We particularly used this technique to generate oxidative DNA damage in the nucleoplasm and in the nucleolus to analyse the possible involvment of two NER initiating factors (CSB and XPC) in oxidative DNA damage repair.
We could clearly establish functionnal recruitment of these factors to our local oxidative DNA damage. The absence of detectable accumulation of downstream NER factors at the local oxidative DNA damage provide the first in vivo indication of the involvement of CSB and XPC in the repair of oxidative DNA lesions independent of the remainder of NER. Interestingly, CSB exhibited different and transcription-dependent kinetics in the two compartments studied (nucleolus and nucleoplasm), suggesting a direct transcription-dependent involvement of CSB in the repair of oxidative lesions associated with different RNA polymerases, but not involving other NER proteins.
Also the recruitment of BER factors like XRCC1 and OGG1 have been investigated and has lead to novel findings. The recruitment of OGG1 to 8-oxoG is not dependant on the presence of CSB; however, in absence of CSB and PARP activity no XRCC1 can be recruited to 8-oxoG. XRCC1 is a very important scaffold protein to allow efficient BER in vivo. The importance of XRCC1 for life is illustrated by the very early embryonic lethality of mice deficient for XRCC1 (Tebbs et al., 1999).
Our results led to a better understanding of the molecular mechanisms of oxidative base damage repair in vivo. The endogenously generated ROS can damage DNA in every organ and probably, more intensively in tissue with high oxygen consumption, like the brain. Our novel findings showing the possible redundancy of PARP and CSB for the efficient loading of XRCC1 may have a major impact to better counteract the deleterious effect of ageing, and oxidative DNA damage on the brain function in the elderly.
David, S.S. V.L. O'Shea, and S. Kundu. 2007. Base-excision repair of oxidative DNA damage. Nature. 447:941 - 50.
Hoeijmakers, J.H. 2001. Genome maintenance mechanisms for preventing cancer. Nature. 411:366 - 74.
Misteli, T., and E. Soutoglou. 2009. The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol. 10:243 - 54.
Tebbs, R.S. M.L. Flannery, J.J. Meneses, A. Hartmann, J.D. Tucker, L.H. Thompson, J.E. Cleaver, and R.A. Pedersen. 1999. Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. Dev Biol. 208:513 - 29