Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

ABSTRACT: Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in real time the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.3 ± 0.4 and 2.0 ± 0.1 nucleotides (nt) s-1 for MGME1 in the 5'-to-3' and 3'-to-5' directions, respectively. These results provide direct evidence of the stronger 5' directionality of MGME1, consistent with its established role in mitochondrial DNA maintenance. Importantly, when we employed NISP to investigate

SUBMITTER: Chiu HP 

PROVIDER: S-EPMC11551736 | biostudies-literature | 2024 Nov

REPOSITORIES: biostudies-literature