SH002 - Bacterial DNA Nucleotide Excision Repair: a multi-approach investigation of the damage recognition process

SH002

Bacterial DNA Nucleotide Excision Repair: a multi-approach investigation of the damage recognition process

M. Genta^1,*, X. Seymonson², A. Biebricher ², D. Jeruzalmi³, A. Chaves⁴, G. Wuite², R. Miggiano¹

¹ Pharmaceutical Sciences , University of Piemonte Orientale, Novara, Italy, ²Physics of Living Systems, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, ³Department of Chemistry and Biochemistry, The City College of New York, Ney York, United States, ⁴Department of Biosciences, University of Milan, Milano, Italy

 

Rationale: Nucleotide Excision Repair (NER) represents one of the major molecular machineries that control chromosome stability in all living species. In Eubacteria, this pathway is carried out by the UvrABC excinuclease complex, comprising the UvrA, UvrB and UvrC proteins. Despite extensive study, NER pathway still lacks a full understanding, and many molecular details about the initial damage recognition process remain controversial. 

Methods: In the past years, we carried out a Cryo-EM-based structural characterization of different complexes between UvrA, UvrB and damaged DNA; to then validate our structural observation, biochemical and biophysical investigations were performed. Recently, we approached optical tweezers in combination with single-molecule fluorescence imaging to determine the molecular forces acting in the damage identification process. 

Results: The structural results have been recently published[1], describing a novel mechanism of damage recognition, in which we revealed that the DNA lesion is first recognized by UvrA thanks to melting, unwinding and flipping-out mechanisms; then, through conformational changes and rearrangements, one UvrB molecule at a time is recruited, to then lead to the pre-incision complex formation, where one UvrB molecule only remains bound on the DNA, with the final aim to recruit UvrC. Recently, we collected new and interesting data about the molecular forces that UvrA applies to the DNA, using optical tweezers: from our preliminary data, it is possible to distinguish different behaviors that UvrA has when interacting with different types of DNA damage (e.g., UV-damaged and Cisplatin-damaged DNA).

 

Conclusions: We are currently working on confirming the preliminary data we collected with optical tweezers. Together with the biochemical and structural characterization, the description of the molecular forces involved in damage recognition will widen the understanding of one of the major bacterial systems.

References: 1 Genta M et al. (2025) Nat Commun 16, (1):3416

Disclosure of Interest: None declared