{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T17:05:39Z","timestamp":1790701539328,"version":"4.1.0"},"reference-count":24,"publisher":"IMR Press","issue":"9","license":[{"start":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:00:00Z","timestamp":1789689600000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Scientific project of the state assignment of Lomonosov Moscow State University","id":[{"id":"https:\/\/ror.org\/010pmpe69","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Front. Biosci. (Landmark Ed)"],"abstract":"<jats:p>Background: Neisseria gonorrhoeae is a human pathogen that causes gonorrhea and employs a methylation-independent mismatch repair (MMR) pathway to regulate genomic stability and pilin antigenic variation. A key effector of this pathway, MutL, possesses ATPase, DNA-binding, and endonuclease activities, yet the molecular determinants governing its catalytic activity remain poorly understood. In particular, the functional relevance of conserved cysteine (Cys) residues located in the vicinity of the active site is unclear. Methods: We characterized a Cys free-variant of Neisseria gonorrhoeae MutL, in which all Cys residues were replaced with evolutionarily conserved substitutes, in comparison with the wild-type enzyme. ATPase activity, DNA-binding capacity, and endonuclease activity on supercoiled plasmid and linear DNA duplex substrates were assessed, and molecular dynamics simulations were performed to examine the conformational behavior of the C-terminal domain (CTD). Results: Cys substitutions do not markedly affect ATPase activity, DNA-binding capacity, or endonuclease activity on supercoiled plasmid substrates. However, the same amino acid substitutions led to an almost complete loss of sliding clamp-dependent endonuclease activity on linear DNA duplexes. Molecular dynamics simulations revealed that the cysteine substitutions alter the conformational dynamics of the CTD, displacing the catalytically active monomer from its productive orientation relative to the sliding clamp and thereby impairing sliding clamp-dependent endonuclease activity. Conclusions: These results indicate that Cys residues are not directly involved in catalysis but provide the conformational rigidity of the C-terminal domain required for productive engagement with the sliding clamp.<\/jats:p>","DOI":"10.31083\/fbl55117","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T16:21:51Z","timestamp":1790698911000},"source":"Crossref","is-referenced-by-count":0,"title":["Cysteine Residues Are Required for the Sliding Clamp-Dependent Endonuclease Activity of Neisseria gonorrhoeae MutL"],"prefix":"10.31083","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-2349-6789","authenticated-orcid":true,"given":"Viktoriia Yu.","family":"Savitskaya","sequence":"first","affiliation":[{"name":"Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2582-6226","authenticated-orcid":true,"given":"Mayya V.","family":"Monakhova","sequence":"additional","affiliation":[{"name":"Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9525-3275","authenticated-orcid":true,"given":"Daniil R.","family":"Bazanov","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasiia V.","family":"Litvinova","sequence":"additional","affiliation":[{"name":"Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8756-5544","authenticated-orcid":true,"given":"Viktoriia G.","family":"Snyga","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2262-117X","authenticated-orcid":true,"given":"Tatiana S.","family":"Oretskaya","sequence":"additional","affiliation":[{"name":"Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-6651-6223","authenticated-orcid":true,"given":"Elena A.","family":"Kubareva","sequence":"additional","affiliation":[{"name":"Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"16242","published-online":{"date-parts":[[2026,9,18]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","unstructured":"Nirwal S, Jha R, Narayanan N, Sharma M, Kulkarni DS, Sharma D, et al. The structure of the MutL-CTD:processivity-clamp complex provides insight regarding strand discrimination in non-methyl-directed DNA mismatch repair. Nucleic Acids Research. 2025; 53: gkaf094.","DOI":"10.1093\/nar\/gkaf094"},{"key":"ref2","doi-asserted-by":"publisher","unstructured":"Hill SA, Masters TL, Wachter J. Gonorrhea - an evolving disease of the new millennium. Microbial Cell (Graz, Austria). 2016; 3: 371\u2013389.","DOI":"10.15698\/mic2016.09.524"},{"key":"ref3","doi-asserted-by":"publisher","unstructured":"Cahoon LA, Seifert HS. Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria. Molecular Microbiology. 2011; 81: 1136\u20131143.","DOI":"10.1111\/j.1365-2958.2011.07773.x"},{"key":"ref4","doi-asserted-by":"publisher","unstructured":"Criss AK, Bonney KM, Chang RA, Duffin PM, LeCuyer BE, Seifert HS. Mismatch correction modulates mutation frequency and pilus phase and antigenic variation in Neisseria gonorrhoeae. 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International Journal of Molecular Sciences. 2023; 24: 6167.","DOI":"10.3390\/ijms24076167"},{"key":"ref8","doi-asserted-by":"publisher","unstructured":"Duppatla V, Bodda C, Urbanke C, Friedhoff P, Rao DN. The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL. The Biochemical Journal. 2009; 423: 265\u2013277.","DOI":"10.1042\/BJ20090626"},{"key":"ref9","doi-asserted-by":"publisher","unstructured":"Namadurai S, Jain D, Kulkarni DS, Tabib CR, Friedhoff P, Rao DN, et al. The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer. PloS One. 2010; 5: e13726.","DOI":"10.1371\/journal.pone.0013726"},{"key":"ref10","doi-asserted-by":"publisher","unstructured":"Guarn\u00e9 A, Charbonnier JB. Insights from a decade of biophysical studies on MutL: Roles in strand discrimination and mismatch removal. 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G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions. Biomedicines. 2022; 10: 1871.","DOI":"10.3390\/biomedicines10081871"},{"key":"ref14","doi-asserted-by":"publisher","unstructured":"Fukui K, Nishida M, Nakagawa N, Masui R, Kuramitsu S. Bound nucleotide controls the endonuclease activity of mismatch repair enzyme MutL. The Journal of Biological Chemistry. 2008; 283: 12136\u201312145.","DOI":"10.1074\/jbc.M800110200"},{"key":"ref15","doi-asserted-by":"publisher","unstructured":"Kosinski J, Plotz G, Guarn\u00e9 A, Bujnicki JM, Friedhoff P. The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron-dependent repressor protein family. Journal of Molecular Biology. 2008; 382: 610\u2013627.","DOI":"10.1016\/j.jmb.2008.06.056"},{"key":"ref16","doi-asserted-by":"publisher","unstructured":"Laitaoja M, Valjakka J, J\u00e4nis J. Zinc coordination spheres in protein structures. 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