Bury Charles, Garman Elspeth F, Ginn Helen Mary, Ravelli Raimond B G, Carmichael Ian, Kneale Geoff, McGeehan John E
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Institute of Nanoscopy, Maastricht University, PO Box 616, Maastricht 6200 MD, The Netherlands.
J Synchrotron Radiat. 2015 Mar;22(2):213-24. doi: 10.1107/S1600577514026289. Epub 2015 Jan 30.
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.
近年来，在从结晶蛋白质收集衍射数据时，大分子晶体学在理解和减轻X射线诱导的辐射损伤方面取得了重大进展。相比之下，尽管有大量研究致力于核酸，特别是DNA的辐射化学领域，但目前关于核酸辐射损伤机制的X射线晶体学研究却很少。分别对蛋白质晶体和DNA晶体的损伤进行定量比较具有挑战性，但通过研究预先形成的生物核蛋白复合物可以规避许多问题，在这种复合物中，可以在相同的受控条件下对每个组分进行直接比较。本文采用一种模型蛋白-DNA复合物C.Esp1396I，在较大剂量范围（2.07-44.63 MGy）内研究生物相关复合物中蛋白质和DNA的特定损伤机制。为了能够对一系列复杂的大分子衍射数据中的辐射损伤位点进行定量分析，开发了一种普遍适用于该领域的计算方法。在特定氨基酸上的蛋白质以及例如碱基-糖N1-C和糖-磷酸C-O键的断裂等DNA上都观察到了典型的特定损伤。引人注目的是，在所研究的剂量范围内，DNA组分被确定比蛋白质对特定损伤的耐受性要强得多。在低剂量时，观察到蛋白质易受辐射损伤，而DNA的耐受性要强得多，只有在显著更高的剂量下才会观察到损伤。
