Radiation damage to nucleoprotein complexes in macromolecular crystallography.

Charles Bury,Raimond B G Ravelli,Geoff Kneale,John E Mcgeehan,Ian Carmichael,Elspeth F Garman,Helen Mary Ginn

doi:10.1107/s1600577514026289

Abstract

Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein-DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07-44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1-C and sugar-phosphate C-O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.

Highlights

Since the advent of powerful third-generation synchrotron sources, significant progress has been made in the field of X-ray crystallography regarding the analysis of X-ray induced radiation damage to proteins during both 100 K and room temperature (RT) diffraction data collection
By using an innovative highly streamlined and automated pipeline for the identification of X-ray induced structural damage patterns, we have established the existence of differential specific damage rates between the protein and DNA components of a model complex C.Esp1396I with respect to dose at 100 K
Whereas other work has studied specific protein and DNA damage in isolation (Spotheim-Maurizot & Davıdkova, 2011; McGeehan et al, 2007; Simons, 2006; Weik et al, 2000; Ravelli & McSweeney, 2000; Burmeister, 2000; Cadet et al, 1999), this work investigated a large dataset of specific damage sites within a protein–DNA complex in order to produce statistically significant observations on specific damage dynamics

Summary

Introduction

Since the advent of powerful third-generation synchrotron sources, significant progress has been made in the field of X-ray crystallography regarding the analysis of X-ray induced radiation damage to proteins during both 100 K and room temperature (RT) diffraction data collection. A wealth of radiation damage studies for nucleic acids has been provided by a strong community of radiation chemists, and mechanisms have been deduced from experiments on individual nucleotides in isolation through to irradiation of whole cells and tissues. The latter studies underpin the development of current radiotherapies in the treatment of a range of cancers, but the full mechanistic X-ray damage landscape from atoms to organisms is far from complete. The atomic resolution that can be provided by X-ray crystallography has great potential to help link the fields of radiation chemistry and radiation biology by providing an atomistic view of radiation damage to intact biological complexes, those involving nucleoproteins

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