The relationship between mtDNA damage sensing by TFAM and selective nucleoid removal in response to environmental exposures

About This Grant

ABSTRACT/PROJECT SUMMARY Transcription factor A mitochondrial (TFAM) has canonical roles in transcription and replication of mitochondrial DNA (mtDNA). Our laboratory has compelling preliminary data that TFAM also has a role in sensing mtDNA lesions caused by UV radiation, resulting in compaction of the mtDNA molecule and its associated proteins, or nucleoid. Because translesion synthesis of replicative machinery past DNA lesions has the capacity to fix them into mutations, a compact nucleoid shape may have the beneficial function of excluding replication machinery and repressing replication. In Aim 1, I will use stimulated emission depletion (STED) microscopy to quantify nucleoid size and colocalization with markers of mtDNA replication and UV damage. This will allow me to test if nucleoids with more damage are more compact in vivo and if they are excluded from replication. Additionally, the compact shape or altered TFAM binding could recruit other proteins to trigger a hitherto unknown process of selective removal from mitochondria. The protein ATAD3A is of particular interest because it binds to both mtDNA and to TFAM and it spans both the outer and inner mitochondrial membranes. To address the hypothesis of selective removal, Aim 2 will test whether knockdown of ATAD3A impairs mtDNA damage removal, and using immunofluorescence, determine the role of ATAD3A in selective localization of damaged nucleoids with lysosomes. To uncover other, unknown protein interactors, Aim 3 will use proximity biotinylation to comprehensively characterize damage-induced changes to the nucleoid proteome. This will be the first description of how nucleoid composition changes after UV and the most temporally-resolved description of changes after environmentally-induced mtDNA damage. Thus, this will be a novel and important contribution to our fundamental understanding of mitochondrial biology. In addition, by identifying proteins that increase in frequency surrounding TFAM after exposure to UV, this technique may identify novel candidates that can be tested for roles in nucleoid compaction and removal. Overall, elucidation of this novel mechanism of mtDNA damage removal will enable future research to identify individuals deficient in this process who may be more susceptible to the harmful effects of exogenous mtDNA damage, with potential benefits in the areas of neurodegeneration. It may also permit development of protective or therapeutic strategies.