Final Report Summary - BERINDR (The role of Base Excision Repair (BER) for extreme radiation and desiccation resistance of Deinococcus radiodurans)
Deinococcus radiodurans (DEIRA) is a pigmented pink/orange bacterium which was first identified in 1956 in canned meat sterilised by ionising radiation. DEIRA exhibits an outstanding resistance to ionising radiation and desiccation and tolerates radiation doses up to 5,000 Grays (Gy) without loss of viability whereas most other organisms cannot survive doses above 50 Gy. Such a massive radiation dose is estimated to induce several hundred double-strand breaks and thousands of single-strand gaps in DNA and about one thousand sites of DNA base damage per chromosome. The resistance mechanism of DEIRA is not known, but initial investigations have suggested that it is complex and most likely determined by a combination of factors such as genome packing, cell structure and highly efficient DNA repair machinery.
The aim of this project was to elucidate the role of the Base Excision Repair (BER) pathway for the extreme radiation and desiccation resistance of the bacterium Deinococcus radiodurans (DEIRA). The BER pathway is highly conserved from bacteria to man and its primary role is to repair endogenous DNA damages like deamination, alkylation, oxidation and single-strand breaks. The recent finding that BER enzymes are central in prevention of double strand breaks suggest that the BER pathway is more important for radiation resistance of DEIRA than previously believed.
Ionising radiation damages DNA by direct ionisation and generates hydroxyl radicals which attack DNA and results in single strand breaks (SSB) and oxidative damages on bases in DNA which later can be converted into double strand breaks (DSB). DNA glycosylases in the BER pathway with specificity for oxidation damages of DNA in bacteria are Endonuclease III (EndoIII), A/G specific adenine DNA glycosylase (MutY) and formamidopyrimidine DNA glycosylase (MutM). Curiously DEIRA possesses three genes encoding Endonuclease III enzymes; DR_2438 (DrEndoIII1), DR_0289 (DrEndoIII2) and DR_0928 (DrEndoIII3), and these three enzymes have been the main targets of study in this project.
Endonuclease III (EndoIII) is a bifunctional DNA glycosylase which repairs a range of oxidised bases in DNA and makes a single strand break in DNA after removal of the damaged base. The enzyme possesses an iron-sulfur cluster [4Fe-4S], and one part of this project was to study the role of this cluster for recognition and removal of damaged DNA bases in DEIRA. Results from electrochemical and spectroscopic studies of DrEndoIII2 strongly indicate that the iron-sulfur cluster is involved in the search and recognition process of damaged bases in DNA, and suggest that the current model for the function of the cluster should be revised. Thus, the results obtained affect the current understanding of the role of the cluster and is of high importance within the field of DNA repair research. One of the most fundamental questions which still remain to be answered in this field is how the DNA glycosylases scan for and recognise damaged bases in DNA. Here we have shed some more light on this question and in a long term perspective the results may be of importance for development of treatments for cancer and neurological diseases.
Structural and catalytic study of the three EndoIII enzymes in DEIRA demonstrated that the enzymes have similar structures, but different substrate specificities. The fellow proposes that the presence of the three EndoIII enzymes in DEIRA extends its DNA repair repertoire and optimise its ability to maintain its genome under exposure to radiation and drought.
As a result of the work performed in this project, one manuscript has been submitted and is under review in the high impact factor international peer reviewed journal JACS (see visual abstract illustration below). Two other papers, describing the crystallisation of DrEndoIII1 and 3, and the structure function study of all three EndoIII proteins are submitted to Acta Crystallographica F and the well acknowledge Journal of Molecular Biology (JMB). A fourth paper, describing the substrate specificity analysis of the EndoIII proteins is in progress, and will be submitted to Plos One. The fellow has recently obtained a post doc fellowship by Fundação para a Ciência e a Tecnologia (FCT) in Portugal, which is a follow up project of the Marie Curie fellowship. The fellow has reported about the progress of the project to the general public in a blog (http://elinsscience.blogspot.pt/(opens in new window)).
The fellow concludes that the project has contributed to increase our understanding of the role of the BER pathway for the radiation and desiccation resistance of DEIRA and underlying mechanisms of DNA repair. The fellow has benefitted immensely from working in a multidisciplinary research institute, and has through the electrochemistry and spectroscopy experiments extended her toolbox within protein function and structure analysis and provided her with important complementary skills, which has enabled her to obtain professional maturity.
The aim of this project was to elucidate the role of the Base Excision Repair (BER) pathway for the extreme radiation and desiccation resistance of the bacterium Deinococcus radiodurans (DEIRA). The BER pathway is highly conserved from bacteria to man and its primary role is to repair endogenous DNA damages like deamination, alkylation, oxidation and single-strand breaks. The recent finding that BER enzymes are central in prevention of double strand breaks suggest that the BER pathway is more important for radiation resistance of DEIRA than previously believed.
Ionising radiation damages DNA by direct ionisation and generates hydroxyl radicals which attack DNA and results in single strand breaks (SSB) and oxidative damages on bases in DNA which later can be converted into double strand breaks (DSB). DNA glycosylases in the BER pathway with specificity for oxidation damages of DNA in bacteria are Endonuclease III (EndoIII), A/G specific adenine DNA glycosylase (MutY) and formamidopyrimidine DNA glycosylase (MutM). Curiously DEIRA possesses three genes encoding Endonuclease III enzymes; DR_2438 (DrEndoIII1), DR_0289 (DrEndoIII2) and DR_0928 (DrEndoIII3), and these three enzymes have been the main targets of study in this project.
Endonuclease III (EndoIII) is a bifunctional DNA glycosylase which repairs a range of oxidised bases in DNA and makes a single strand break in DNA after removal of the damaged base. The enzyme possesses an iron-sulfur cluster [4Fe-4S], and one part of this project was to study the role of this cluster for recognition and removal of damaged DNA bases in DEIRA. Results from electrochemical and spectroscopic studies of DrEndoIII2 strongly indicate that the iron-sulfur cluster is involved in the search and recognition process of damaged bases in DNA, and suggest that the current model for the function of the cluster should be revised. Thus, the results obtained affect the current understanding of the role of the cluster and is of high importance within the field of DNA repair research. One of the most fundamental questions which still remain to be answered in this field is how the DNA glycosylases scan for and recognise damaged bases in DNA. Here we have shed some more light on this question and in a long term perspective the results may be of importance for development of treatments for cancer and neurological diseases.
Structural and catalytic study of the three EndoIII enzymes in DEIRA demonstrated that the enzymes have similar structures, but different substrate specificities. The fellow proposes that the presence of the three EndoIII enzymes in DEIRA extends its DNA repair repertoire and optimise its ability to maintain its genome under exposure to radiation and drought.
As a result of the work performed in this project, one manuscript has been submitted and is under review in the high impact factor international peer reviewed journal JACS (see visual abstract illustration below). Two other papers, describing the crystallisation of DrEndoIII1 and 3, and the structure function study of all three EndoIII proteins are submitted to Acta Crystallographica F and the well acknowledge Journal of Molecular Biology (JMB). A fourth paper, describing the substrate specificity analysis of the EndoIII proteins is in progress, and will be submitted to Plos One. The fellow has recently obtained a post doc fellowship by Fundação para a Ciência e a Tecnologia (FCT) in Portugal, which is a follow up project of the Marie Curie fellowship. The fellow has reported about the progress of the project to the general public in a blog (http://elinsscience.blogspot.pt/(opens in new window)).
The fellow concludes that the project has contributed to increase our understanding of the role of the BER pathway for the radiation and desiccation resistance of DEIRA and underlying mechanisms of DNA repair. The fellow has benefitted immensely from working in a multidisciplinary research institute, and has through the electrochemistry and spectroscopy experiments extended her toolbox within protein function and structure analysis and provided her with important complementary skills, which has enabled her to obtain professional maturity.