Mitochondria contain their own genome encoding a portion of the oxidative phosphorylation system responsible for ATP synthesis required for cellular function, and as such, also contain a conserved system for maintaining genomic integrity. Current biochemical studies demonstrate that maintenance of the mitochondrial genome at degradation stages are performed by the same machinery used for replication. A DNAse, MGME1, has been identified in directly aiding these processes as a complementary nuclease for the lack of 5’ to 3’ nuclease activity in mitochondrial DNA polymerase γ, but has yet had biochemical confirmation to support this function. Herein we provide clear, quantitative evidence that MGME1 directionality is predominantly in the 5’ to 3’ direction. We find that MGME1 binding and activity depends on the presence of a 5’ phosphate group and also is influenced by the DNA sequence.<br/><br/>Key binding information on MGME1 in the presence of single-stranded DNA (ssDNA) homopolymers containing either a 3’- or 5’-fluorescein (FAM) labeled end has established that MGME1 preferably binds to ssDNA that can accommodate a 10-nucleotide binding footprint. Compared to ssDNA bearing a hydroxyl group at the 3’- or 5’-end, increased exonuclease activity is observed with a free 5’-end containing a phosphate group, showing a clear preference for the 5’-end. We have also found sequence preferences using 5’-FAM labeled random-sequence DNA and poly d(T20), d(A20), and d(C20) oligomers, where thymine-rich DNA is found to be the fastest degraded in comparison to DNA containing adenine or cytosine.<br/><br/>In summary, we report quantitative DNA-binding and kinetic data that clearly define the directionality of MGME1 that has been controversial in literature. Although MGME1 lacks a phosphate-binding pocket, several positively-charged motifs within the N-terminus and C-terminus domains may facilitate interactions with the phosphate via charge-charge interactions and hydrogen bonding. Further investigation is needed to determine the roles of these motifs in interacting with the terminal phosphate.

Preferential binding and cleavage from 5'-ends for human mitochondrial genome maintenance nuclease 1 (MGME1) is conferred by a phosphate group

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