TY - JOUR
T1 - Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage
AU - Alduina, Rosa
AU - Tagliaferri, Sara
AU - Mancini, Monica
AU - Castani, Federica
AU - Ferrandi, Alex
AU - Pitaro, Mauro
AU - Tagliaferri, Sara
AU - Tagliaferri, Sara
AU - De La Tour, Claire Bouthier
AU - Bonapace, Ian Marc
AU - De La Tour, Claire Bouthier
AU - Fasano, Mauro
AU - Barbieri, Paola
AU - Sommer, Suzanne
PY - 2019
Y1 - 2019
N2 - Background: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes.Methods: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coif and DR. slip knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette.Results: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm(+) genomic DNA, and to cytosine unmethylated DR and E. coli dcm(-) genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced > 100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors.Conclusions: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage.General significance: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.
AB - Background: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes.Methods: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coif and DR. slip knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette.Results: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm(+) genomic DNA, and to cytosine unmethylated DR and E. coli dcm(-) genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced > 100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors.Conclusions: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage.General significance: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.
UR - http://hdl.handle.net/10447/301900
M3 - Article
VL - 1863
SP - 118
EP - 129
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0006-3002
ER -