Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single-molecule studies

SignificanceWe directly visualize DNA translocation and lesion recognition by the O -alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn occupancy of AGT. A role of cooperative AGT clusters in enhancing lesio...

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Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 119; no. 11; p. e2116218119
Main Authors:	Kono, Sarah, van den Berg, Aafke, Simonetta, Marco, Mukhortava, Ann, Garman, Elspeth F, Tessmer, Ingrid
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 15-03-2022
Subjects:	Alkyl and Aryl Transferases - chemistry Alkyl and Aryl Transferases - metabolism Atomic force microscopy Biological Sciences Chemotherapy Clusters Crystal structure Cytotoxicity Deoxyribonucleic acid DNA DNA - chemistry DNA - genetics DNA - metabolism DNA alkyltransferase DNA Repair DNA, Single-Stranded Fluorescence Fluorescence microscopy Humans Ions Lesions Microscopy Models, Molecular O-Methylguanine-DNA Methyltransferase - chemistry O-Methylguanine-DNA Methyltransferase - metabolism Protein Multimerization Proteins Repair Searching Single Molecule Imaging - methods Structure-Activity Relationship Substrates Translocation Zinc - chemistry fluorescence optical tweezers DNA alkyltransferase DNA repair single-molecule studies
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Summary:	SignificanceWe directly visualize DNA translocation and lesion recognition by the O -alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by Eric Greene, Columbia University, New York, NY; received September 2, 2021; accepted February 8, 2022 by Editorial Board Member Rodney Rothstein Author contributions: I.T. designed research; S.K., E.F.G., and I.T. performed research; A.v.d.B., M.S., A.M., and E.F.G. contributed new reagents/analytic tools; S.K., E.F.G., and I.T. analyzed data; and I.T. wrote the paper.
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.2116218119