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

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.

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