Biochemical characterization of mammalian NEIL3 and involvement in repair of hydantoin lesions in proliferative tissue.
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AbstractOxidative damage is a major threat to the integrity of our genome and can lead to mutations or block replication if not repaired. Base excision repair (BER) is the main pathway for repair of oxidative lesions and is initiated by the action of a DNA glycosylase. A monofunctional glycosylase recognizes and excises the modified nucleotide, while a bifunctional glycosylase in addition possesses an intrinsic apurinic/apyrimidinic (AP) lyase activity generating a nick in the DNA strand. There are five DNA glycosylases acting on oxidative damage in eukaryotic cells; 8-oxoguanine DNA glycosylase (OGG1), endonuclease III (NTH1), and endonuclease VIII-like 1, 2 and 3 (NEIL1, NEIL2 and NEIL3). These enzymes are bifunctional with overlapping substrate specificity and knockout mouse studies reveals that none of them are essential for life. NEIL3 is sequentially related to the well characterized enzymes NEIL1 and 2, but less is known about its biochemical properties. Paper I of this thesis includes an expression and purification protocol for human NEIL3. NEIL3 recognizes and excises guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from the genome. In paper II we elucidate the amino acids important for activity. NEIL1 and NEIL2 have tightly coupled DNA glycosylase/AP lyase activities and perform β,δ-elimination mechanism to incise DNA. NEIL3 has uncoupled activities, where the base excision is more efficient than the strand incision. We demonstrate that a V2P mutation changes the catalytic properties of NEIL3 to be similar to the activity of NEIL1 and 2. While OGG1, NTH1, NEIL1 and NEIL2 are expressed in most tissues throughout the organism, NEIL3 expression is limited to some lymphatic organs and compartments of the brain harboring neural stem cells, indicating a role in highly proliferative tissue. Paper III shows that mouse Neil3 is responsible for repair of Gh and Sp lesions in single-stranded DNA (ssDNA) in thymus and spleen, where repair activity correlates with Neil3 expression level. In paper IV of this study, we expose young Neil3- deficient mice to hypoxia-ischemia, to introduce cell death and activate proliferation of neural tissue. We find that the Neil3 deficit leads to reduced regeneration of neural tissue in the striatum. Further, in vitro propagated Neil3-deficient neural stem cells have a depressed neurogenesis and impaired ability to repair Gh and Sp lesions in ssDNA. Taken together, we suggest that NEIL3 has a unique role in removing hydantoin lesions from ssDNA and thereby promoting replication of proliferating cells.
List of papers. The papers are removed from the thesis due to publisher restrictions.
Paper I: Silje Z. Krokeide, Nils Bolstad, Jon K. Laerdahl, Magnar Bjørås and Luisa Luna. Expression and purification of NEIL3, a human DNA glycosylase homolog. Protein Expression and Purification 2009 Jun;65(2):160-4. doi:10.1016/j.pep.2008.11.014
Paper II: Silje Z. Krokeide, Medya Salah, Jon K. Laerdahl, Luisa Luna, F. Henning Cederkvist, Aaron M. Fleming, Cynthia J. Burrows, Bjørn Dalhus, and Magnar Bjørås. Human NEIL3 is mainly a monofunctional DNA glycosylase with affinity to spiroiminodihydantoin and guanidinohydantoin. DNA Repair 2013 Jun 5 [Epub ahead of print] doi:10.1016/j.dnarep.2013.04.026
Paper III: Veslemøy Rolseth, Silje Zandstra Krokeide, David Kunke, Christine Gran Neurauter, Rajikala Suganthan, Yngve Sejersted, Gunn Annette Hildrestrand, Magnar Bjørås and Luisa Luna. Loss of Neil3, the major DNA glycosylase activity for removal of hydantoins in single-stranded DNA, reduces cellular proliferation and sensitizes cells to genotoxic stress. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2013 May;1833(5):1157-64. doi:10.1016/j.bbamcr.2012.12.024
Paper IV: Yngve Sejersted, Gunn Annette Hildrestrand, David Kunke, Veslemøy Rolseth, Silje Zandstra Krokeide, Christine Gran Neurauter, Rajikala Suganthan, Monica AtneosenÅsegg, Aaron M. Fleming, Ola Didrik Saugstad, Cynthia J. Burrows, Luisa Luna, and Magnar Bjørås. Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia. Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2011 Nov15;108(46): 18802-7. doi:10.1073/pnas.1106880108