Aamann, Maria Diget4; Hvitby, Christina Poulsen5; Popuri, Venkateswarlu6; Muftuoglu, Meltem10; Lemminger, Lasse Pagh Brøgger5; Skeby, Cecilie K7; Keijzers, Guido10; Ahn, Byungchan8; Bjørås, Magnar9; Bohr, Vilhelm A3; Stevnsner, Tinna V.4
1 Section I. Center for Healthy Aging, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet2 Center for Healthy Ageing, Faculty of Health and Medical Sciences, Københavns Universitet3 Molecular Aging Program, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet4 Institut for Molekylærbiologi og Genetik - Genomekspression, stabilitet og teknologi5 Molekylærbiologisk Institut6 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA.7 Danish Center for Molecular Gerontology and Danish Aging Research Center, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.8 Institut for Molekylærbiologi og Genetik9 Laboratory for Molecular Biology, Center for Molecular Biology and Neuroscience, Oslo University Hospital, Oslo, Norway.10 Section I. Center for Healthy Aging, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet
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