Investigation of strategies and synergies in DNA lesion recognition using single molecule AFM imaging

• Applicant: Ingrid Teßmer, Ph.D.
• Subject Area: Biophysics ; Biochemistry ; Structural Biology
• Term: from 2014 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 254014264

• Final Report Year: 2022

Final Report Abstract

We directly visualize DNA translocation and lesion recognition by the O6-alkylguanine DNA alkyltransferase, AGT using a combined fluorescence optical tweezers system. From our data we derive a new model for the lesion search and repair mechanism of AGT, in which AGT moves bidirectionally on DNA during lesion search either by slipping in a loosely bound conformation or by rotational movement along the DNA minor groove. Initial lesion recognition by AGT then triggers cluster formation by AGT at the lesion, which further stabilizes the complex with DNA and facilitates complete insertion of the lesion base into the catalytic site pocket of AGT supported by additional strain induced in the DNA due to a slight degree of DNA bending by the additional monomer subunits in the cluster. In the catalytic site pocket, the alkyl group is then transferred from the guanine base onto the catalytic cysteine in AGT to complete the repair process. The additional monomer subunits in the cluster are also available for direct protein-protein interactions for recruitment of proteins, for example from the DNA replication machinery, to allow for rapid replication restart after successful removal of the highly mutagenic alkylguanine lesions by AGT. For the catalytically inactive AGT homolog, the alkyltransferase-like protein, ATL, we have been able to show co-translocation on DNA with the initiating enzyme of (prokaryotic) nucleotide excision repair (NER), UvrA. ATL itself cannot repair alkyl lesions in DNA, but has evolved to mark these lesions and, as we showed in fluorescence optical tweezers, atomic force microscopy (AFM), and biochemical analyses, recruit the NER system to repair the alkyl lesions that are by themselves not native targets of NER. We further applied single molecule AFM imaging to investigate a range of base excision repair (BER) glycosylases with structurally different target lesions, as well as these DNA lesions in the absence of protein. Using a novel, automated, unbiased, high-throughput analysis approach, which we developed for these studies, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search that were tailored to the mechanical properties of their respective target lesions. Our results thus lent support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions.

Publications

• (2014) Insight into the cooperative DNA binding of the O6- alkylguanine DNA alkyltransferase, special issue on “Single molecule approaches: watching DNA repair one molecule at a time”, DNA Repair 20: 14-22
  I Tessmer and MG Fried
  
• (2014) Strand specific recognition of DNA damages by XPD provides insights into Nucleotide Excision Repair substrate versatility, Journal of Biological Chemistry 289(6): 3613-3624
  CN Buechner, K Heil, G Michels, T Carell, C Kisker, I Tessmer
  
• (2015) Characterization of homogeneous, cooperative protein-DNA clusters by sedimentation equilibrium analytical ultracentrifugation and atomic force microscopy, in: Methods in Enzymology 562 “Analytical Ultracentrifugation” (ed. J Cole): 331-348
  I Tessmer and MG Fried
  
• (2015) Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging, Nucleic Acids Research 43(5): 2716-2729
  CN Buechner, A Maiti, AC Drohat, I Tessmer
  
• (2016) Conservation and divergence in nucleotide excision repair, Journal of Biological Chemistry 291(36): 18932-18946
  N Wirth, J Gross, HM Roth, C Buechner, C Kisker, I Tessmer
  
• (2017) Atomic force microscopy investigations of DNA lesion recognition in nucleotide excision repair, Journal of Visualized Experiments (123), e55501
  J Gross, N Wirth, I Tessmer
  
• (2018) Unique insight into protein-DNA interactions from single molecule atomic force microscopy, AIMS Biophysics 5(3): 194-216
  DM Bangalore and I Tessmer
  
• (2020) Alkyltransferase-like protein clusters scan DNA rapidly over long distances and recruit NER to alkyl-DNA lesions, Proceedings of the National Academy of Sciences of the USA 117(17): 9318-9328
  N Rill, A Mukhortava, S Lorenz, I Tessmer
  
• (2020) Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases, Scientific Reports 10(1), 15484
  DM Bangalore, HS Heil, CF Mehringer, L Hirsch, K Hemmen, KG Heinze, I Tessmer
  
• Resolving the subtle details of human DNA alkyltransferase lesion search and repair mechanism by single molecule studies, PNAS 2022 Mar 15;119(11):e2116218119
  S Kono, A van den Berg, M Simonetta, A Mukhortava, EF Garman, I Tessmer