Die vielfältigen Funktionen des Brustkrebs-Suszebilitätsgens 1 (BRCA1) in Pflanzen
Plant Biochemistry and Biophysics
Plant Physiology
Plant Breeding and Plant Pathology
Final Report Abstract
BRCA1 is an important tumor suppressor protein in humans and with its manifold functions it is a major contributor to genome stability. Mutations in HsBRCA1 are associated with genomic instability, and most strikingly an elevated risk of breast cancer. As homozygous BRCA1 mutations in mammals are embryo lethal, in vivo analyses are extremely difficult in these systems. In Arabidopsis thaliana a BRCA1 homolog could be identified and its viable brca1 mutants render it a convenient heterologous model for the analysis of this important protein. With this project we were able to further characterize AtBRCA1 in its function in DNA repair and to clarify its domain functions. We could unveil a function for AtBRCA1 in the repair of CPT-induced DNA damage, thereby resembling its human homolog. Furthermore, using complementation analyses we were able to characterize the different domains of AtBRCA1 in CPT-induced damage repair for the first time. We were able to exclude a participation of the plant specific PHD domain as well as the RING domain in the repair of CPT-induced DNA-protein crosslinks. Interestingly, while the mutation of the serine residue at position 738 in the first BRCT domain of AtBRCA1 did not have any effect on BRCA1 function, our data suggest a crucial role for the lysine residue at position 785 (construct CTM2) in the same domain. Mutant lines containing constructs with both mutations in the BRCT domain showed strong deficiencies in the repair of CPT-induced DNA damage. This implies an important function for the BRCT domain in DNA repair processes occurring after CPT-induced damage. FANCJ helicases participate with BRCA1 in DSB-repair, but are also involved in the Fanconi anemia (FA) pathway of crosslink repair in humans, thus linking these repair mechanisms. We were able to identify two FANCJ homologs in Arabidopsis thaliana, and could show that at least one of them AtFANCJB has an important role in DNA crosslink repair. Furthermore, we could demonstrate functional epistasis between AtBRCA1 and AtFANCJB, resembling its human counterparts. As we could already postulate a model of DNA crosslink repair in Arabidopsis with three distinct subpathways, it was of special interest to classify FANCJB into these branches. While AtFANCJB acts independently of AtMUS81, we were able to demonstrate a cooperation of AtFANCJB with AtRECQ4A and interestingly with AtRAD5A, thereby connecting the FA pathway with post replicative DNA repair. Such a connection has not been shown to our best knowledge for any other multicellular organism before.
