Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions

Abstract

Human mitochondrial DNA (mtDNA) encodes 37 essential genes and plays a critical role in mitochondrial and cellular functions. mtDNA is susceptible to damage by endogenous and exogenous chemicals. Damaged mtDNA molecules are counteracted by the redundancy, repair, and degradation of mtDNA. In response to difficult-to-repair or excessive amounts of DNA lesions, mtDNA degradation is a crucial mitochondrial genome maintenance mechanism. Nevertheless, the molecular basis of mtDNA degradation remains incompletely understood. Recently, mitochondrial transcription factor A (TFAM) has emerged as a factor in degrading damaged mtDNA containing abasic (AP) sites. TFAM has AP-lyase activity, which cleaves DNA at AP sites. Human TFAM and its homologs contain a higher abundance of Glu than that of the proteome. To decipher the role of Glu in TFAM-catalyzed AP-DNA cleavage, we constructed TFAM variants and used biochemical assays, kinetic simulations, and molecular dynamics (MD) simulations to probe the functional importance of E187 near a key residue K186. Our previous studies showed that K186 is a primary residue to cleave AP-DNA via Schiff base chemistry. Here, we demonstrate that E187 facilitates β-elimination, key to AP-DNA strand scission. MD simulations showed that extrahelical confirmation of the AP lesion and the flexibility of E187 in TFAM-DNA complexes facilitate AP-lyase reactions. Together, highly abundant Lys and Glu residues in TFAM promote AP-DNA strand scission, supporting the role of TFAM in AP-DNA turnover and implying the breadth of this process across different species.

Unprecedented reactivity of polyamines with aldehydic DNA modifications: structural determinants of reactivity, characterization and enzymatic stability of adducts

“…Both AP sites and fU are abundant DNA modifications that occur in the genome at comparable rates ( 46 ). Due to their aldehyde nature, they are prone to reactions with nucleophiles; however, until recently, the main reactivity of AP sites was considered to be due to the reversible formation of Schiff bases with lysine residues of proteins ( 21–23 ). Only recently other types of AP site reactivity were discovered, namely the formation of thiazolidines ( 19 , 20 ) and S -glycosylic derivatives ( 17 , 18 ) upon reaction with cysteine residues, as well as nucleophilic addition of exocyclic amino groups of neighboring nucleobases leading to the formation of N -glycosylic DNA ICLs ( 9–14 ).…”

35 Years of TFAM Research: Old Protein, New Puzzles

“…This processing includes AP incision with abundant AP endonuclease (APE1) ‘5 to the lesion and the removal of the resulting 5’ deoxyribosephosphate (dRP) group by DNA polymerase γ [ 130 ]. Recent data suggest that TFAM stimulates the incision of AP sites in mtDNA, reducing their half-life by 2–3 orders of magnitude to minutes, thus promoting their repair [ 131 , 132 , 133 , 134 , 135 ]. Earlier studies revealed preferential TFAM binding to DNA containing oxidatively damaged bases [ 136 ] and to DNA 4-way junctions [ 137 ], but surprisingly, not AP sites [ 136 ].…”