Aamann, Maria Diget10; Hvitby, Christina Poulsen10; Popuri, Venkateswarlu6; Muftuoglu, Meltem7; Lemminger, Lasse Pagh Brøgger3; K. Skeby, Cecilie8; Keijzers, Guido4; Ahn, Byungchan11; Bjørås, Magnar9; Bohr, Vilhelm6; Stevnsner, Tinna V.10
1 Department of Clinical Medicine - Biomedical Radio Isotope Techniques, Department of Clinical Medicine, Health, Aarhus University2 Department of Molecular Biology and Genetics - Genome stability and technology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University3 Department of Molecular Biology and Genetics - Department of Molecular Biology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University4 Department of Molecular Biology, Faculty of Science, Aarhus University, Aarhus University5 Department of Molecular Biology and Genetics, Science and Technology, Aarhus University6 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD7 Center for Healthy Aging, University of Copenhagen8 unknown9 Laboratory for Molecular Biology, Center for Molecular Biology and Neuroscience, Oslo University Hospital, Oslo10 Department of Molecular Biology and Genetics - Genome stability and technology, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University11 Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase activity on a 5-hydroxyl uracil lesion in a DNA bubble structure substrate in vitro. A novel 4,6-diamino-5-formamidopyrimidine (FapyA) specific incision activity of NEIL2 was also stimulated by CSB. To further elucidate the biological role of the interaction, immunofluorescence studies were performed, showing an increase in cytoplasmic CSB and NEIL2 co-localization after oxidative stress. Additionally, stalling of the progression of the transcription bubble with α-amanitin resulted in increased co-localization of CSB and NEIL2. Finally, CSB knockdown resulted in reduced incision of 8-hydroxyguanine in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair.
Mechanisms of Ageing and Development, 2014, Vol 135, p. 1-14
Cockayne Syndrome; CSB; NEIL2; Base excision repair; Oxidative damage