{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"Base Excision DNA Repair in the Mitochondria of Differentiated Neuronal Cells","metadata":[{"label":"dc.format","value":"application/pdf"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/11401/78297"},{"label":"dcterms.abstract","value":"Healthy mitochondria are vital for maintaining neuronal activity over the lifespan of an organism. Since mitochondria contain their own DNA, functional DNA repair pathways in the organelle are necessary for mitochondrial integrity. However, mitochondria do not have the complete complement of DNA repair pathways found in the nucleus. Thus far, base excision DNA repair (BER), mismatch repair, and double-strand break repair have been detected in mitochondria, although these pathways involve fewer proteins than is the case in the nucleus. Of the DNA repair pathways operating in mitochondria, BER acts on small, non-distorting base lesions, the main type of damage expected to arise from oxidative reactions.  This dissertation focuses on BER after cellular differentiation. There is mounting evidence that BER occurs more slowly in differentiated cells compared to proliferating cells. In terminally differentiated cells, the nuclear genome is no longer being replicated, and the proteins involved in DNA replication are correspondingly down-regulated in the nucleus. However, mitochondria continue to replicate their DNA and divide even in a non-dividing cell, which necessitates efficient DNA repair. It may be that mitochondrial DNA repair proteins shared with the nucleus continue to be imported into mitochondria after differentiation. It is also possible that other, mitochondria-specific proteins compensate for the loss of shared DNA repair proteins. Of particular interest are four nuclease enzymes implicated in \u201clong-patch\u201d base excision DNA repair in mitochondria: Fen1, DNA2, MGME1 and ExoG.  During DNA repair synthesis in base excision repair, the DNA polymerase can add a single nucleotide, at which point the enzyme removes the resulting 5'-terminal 2-deoxyribose-5-phosphate (5\u2019-dRP) residue, and DNA ligase III can seal the nick. However, if the DNA polymerase adds more than one nucleotide, the resulting displaced oligonucleotide flap needs to be removed before the DNA strand can be resealed by DNA ligase. That process is so-called \u201clong-patch\u201d BER. The nuclease(s) responsible for flap excision in the mitochondria are not yet certain. Fen1 and DNA2 are enzymes that function in the maturation of Okazaki fragments during DNA replication in the nucleus, as well as in nuclear and mitochondrial DNA repair. ExoG and MGME1 are structure-specific nucleases specific to mitochondria, and they have activities that could function in long-patch BER. Investigating the regulation and roles of these proteins should give insight into the modulation of BER during cellular differentiation.  The use of CAD cells, a mouse neuroblastoma cell line that can be differentiated into neurons in vitro, allows the comparison of proliferating progenitors and genetically identical terminally differentiated cells. Immunoblotting and immunofluorescence microscopy were used to probe the expression of these proteins in the nucleus and in mitochondria. Upon CAD cell differentiation, Fen1 and DNA2 were indeed down-regulated at the whole cell level. ExoG was also lowered to 50% of the cycling cell level, while MGME1 was not diminished and may be modestly increased. Enzyme activity assays showed that whole-cell extracts of differentiated cells performed flap excision less effectively than did the proliferating cell extracts. Likewise, mitochondrial extracts from differentiated CAD cells had slower flap excision than observed for mitochondrial extracts from proliferating cells. Paradoxically, a PCR-based DNA repair assay for mitochondrial DNA damage revealed that differentiated cell mitochondria accumulated less damage than did proliferating cell mitochondria. Taken together, these data imply that long-patch BER flap excision occurs more slowly in differentiated cells, both in the nucleus and mitochondria, but that, despite the difference in the flap endonuclease activity detected, the in vivo mitochondrial DNA repair capacity of differentiated cells is comparable to that of proliferating cell mitochondria."},{"label":"dcterms.available","value":"2020-04-06"},{"label":"dcterms.contributor","value":"Advisors: Demple, Bruce; Tsirka, Styliani-Anna; Bogenhagen, Daniel; Colognato, Holly; Boon, Elizabeth"},{"label":"dcterms.creator","value":"Caston, Rachel Audrey"},{"label":"dcterms.dateAccepted","value":"2018-07-03T17:16:42Z"},{"label":"dcterms.dateSubmitted","value":"2018-07-03T17:16:42Z"},{"label":"dcterms.description","value":"Dissertation"},{"label":"dcterms.extent","value":"115 pages"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/11401/78297"},{"label":"dcterms.issued","value":"2017-12-01"},{"label":"dcterms.language","value":"en"},{"label":"dcterms.provenance","value":"Submitted by Jason Torre (fjason.torre@stonybrook.edu) on 2018-07-03T17:16:42Z\nNo. of bitstreams: 1\nCaston_grad.sunysb_0771E_13596.pdf: 2014449 bytes, checksum: ffbe01014e7bea10d8c6ba1062a3cd4e (MD5)"},{"label":"dcterms.publisher","value":"Stony Brook University"},{"label":"dcterms.subject","value":"Molecular biology, Differentation, DNA Repair, Mitochondria, Reactive Oxygen Species"},{"label":"dcterms.title","value":"Base Excision DNA Repair in the Mitochondria of Differentiated Neuronal Cells"},{"label":"dcterms.type","value":"Text"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/30%2F44%2F54%2F30445408037417810291446891653375237315/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/30%2F44%2F54%2F30445408037417810291446891653375237315","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}