Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA.

Aleksandr Alekseev,Maksim Serdakov,Irina Bakhlanova,Mikhail Khodorkovskii,Galina Cherevatenko,Georgii Pobegalov,Alexander Yakimov,Dmitry Baitin

doi:10.3390/ijms21197389

Abstract

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Highlights

The radioresistant bacterium Deinococcus radiodurans and other members of the Deinococcaceae show an outstanding capacity to cope with high dosage of ionizing radiation and other DNA-damaging agents, such as desiccation, ultraviolet radiation, and diverse genotoxic chemicals [1,2,3,4,5]
To register the assembly of the DrRecA–ssDNA filament, a single ssDNA molecule was introduced to the channel containing 1 μM DrRecA and 1 mM ATP, and the change in the end-to-end distance was monitored under a constant applied tension of 12 pN that facilitated the removal of ssDNA secondary structures
We examined the dynamics of interaction of DrRecA with both ssDNA and double-stranded DNA (dsDNA) using a single-molecule approach

Summary

Introduction

The radioresistant bacterium Deinococcus radiodurans and other members of the Deinococcaceae show an outstanding capacity to cope with high dosage of ionizing radiation and other DNA-damaging agents, such as desiccation, ultraviolet radiation, and diverse genotoxic chemicals [1,2,3,4,5]. The extreme radiation resistance of D. radiodurans has been attributed to a strong protection of the proteins from oxidative damage [6,7] and a very robust DNA repair system [3,8]. DrRecA belongs to a highly conserved family of bacterial homologous recombination proteins that promote the error-free repair of DNA damage [10,11]. D. radiodurans expressing RecA variants defective in recombination are highly sensitive to ionizing radiation [12,13]. Apart from homologous recombination, DrRecA was reported to be crucial for the extended synthesis-dependent strand annealing (ESDSA), which is a unique mechanism of fragmented chromosome segments assembly at the early phase of DNA repair in D. radiodurans [8,14]. D. radiodurans is immutable by ultraviolet radiation due to the error-free reparation of ultraviolet-induced DNA damage [15], whereas an error-prone pathway for the repair of such DNA damage was found in D. deserti [16], which is another species belonging to Deinococcaceae

Methods

Results

Conclusion