Structural basis of how MGME1 processes DNA 5^′ ends to maintain mitochondrial genome integrity

Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5^′-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5^′ or 3^′ end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5^′ end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5^′-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5^′-to-3^′ or 3^′-to-5^′ direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5^′-DNA flaps, providing insights into MGME1-mediated 5^′-end processing of nascent mtDNA. Our study builds on previously solved MGME1–DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.