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Using biophysical, single-molecule and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). In half of events, the partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, looping is more frequent and both motors can engage in bidirectional loop translocation. Cleavage occurs stochastically throughout translocation but events that increase dwell time such as rebinding methylated sites elevate the chance of cleavage. Interactions with other SauUSI dimers or protein roadblocks influence cleavage distributions but are not required for nuclease activation. While the first cleavages principally generate 1-nt 3\u02b9 overhangs or blunt ends, multiple binding\u2013translocation\u2013cleavage cycles by individual enzymes ultimately fragment the modified nonself DNA, neutralizing its threat.\n                  <\/jats:p>","DOI":"10.1038\/s41589-026-02353-4","type":"journal-article","created":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T17:49:13Z","timestamp":1792086553000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single Staphylococcus aureus SauUSI restriction enzyme motors fragment methylated DNA"],"prefix":"10.1038","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1236-1810","authenticated-orcid":false,"given":"Steven J.","family":"Shaw","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9540-040X","authenticated-orcid":false,"given":"Alex","family":"Hughes-Games","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4234-6037","authenticated-orcid":false,"given":"Oscar E.","family":"Torres Montaguth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sam","family":"McDowall","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fiona M.","family":"Diffin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Dunn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3565-0479","authenticated-orcid":false,"given":"Stephen J.","family":"Cross","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2501-6602","authenticated-orcid":false,"given":"Mark D.","family":"Szczelkun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,10,15]]},"reference":[{"key":"2353_CR1","doi-asserted-by":"publisher","DOI":"10.1128\/ecosalplus.esp-0014-2022","volume":"13","author":"BP Anton","year":"2025","unstructured":"Anton, B. 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