Monitoring DNA polymerase β mitochondrial localization and dynamics.

Autor: Horton JK; Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: horton1@niehs.nih.gov., Janoshazi AK; Fluorescence Microscopy and Imaging Center, Signal Transduction Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA., Nadalutti CA; Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA., Zhao ML; Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA., Stefanick DF; Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA., Wilson SH; Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Jazyk: angličtina
Zdroj: DNA repair [DNA Repair (Amst)] 2022 Aug; Vol. 116, pp. 103357. Date of Electronic Publication: 2022 Jun 11.
DOI: 10.1016/j.dnarep.2022.103357
Abstrakt: Mouse fibroblasts lacking (null) DNA polymerase β (pol β) were transfected with fluorescently tagged pol β and stained with biomarkers to allow visualization within living cells by confocal microscopy. Transient transfection resulted in varying pol β expression levels. Separating cells into three groups based on pol β fluorescence intensity and morphological distribution, permitted analysis of the concentration dependence and spatial distribution of cytoplasmic pol β. Colocalization between pol β and mitochondria was pol β concentration dependent. A decrease in overlap with nucleoids containing mitochondrial DNA (mtDNA) was observed at the highest pol β intensity where pol β exhibits a tubular appearance, suggesting the ability to load elevated levels of pol β into mitochondria readily available for relocation to damaged mtDNA. The dynamics of pol β and mitochondrial nucleoids were followed by confocal recording of time series images. Two populations of mitochondrial nucleoids were observed, with and without pol β. Micro-irradiation, known to form DNA single-strand breaks, in a line across nucleus and cytoplasm of pol β stably transfected cells enhanced apparent localization of pol β with mitochondria in the perinuclear region of the cytoplasm near the nuclear membrane. Exposure of pol β expressing cells to H 2 O 2 resulted in a time-dependent increase in cytoplasmic pol β observed by immunofluorescence analysis of fixed cells. Further screening revealed increased levels of colocalization of pol β with a mitochondrial probe and an increase in oxidative DNA damage in the cytoplasm. ELISA quantification confirmed an increase of an oxidative mitochondrial base lesion, 7,8-dihydro-8-oxoguanine, after H 2 O 2 treatment. Taken together, the results suggest that pol β is recruited to mitochondria in response to oxidatively-induced mtDNA damage to participate in mtDNA repair.
(Published by Elsevier B.V.)
Databáze: MEDLINE
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