Structural Biology Study Of SspE Of The Type ? DNA Phosphorothioate Modification System

DNA phosphorothioation(PT)is a new type of modification,which occurs on DNA skeleton by substituting sulfur atoms for non-bridged oxygen atoms on phosphodiester bond.It is regulated by gene cluster dndABCDE and modified in specific sequences on double strands in a specific spatial configuration(Rp),which is discovered in bacteria and archaea.Further studies show that there are reverse restriction gene dndFGH downstream of the modified gene cluster,which together constitute a restriction-modification system to maintain the genetic integrity of host bacteria.With the development of research,another type modification(CpsC),which is responsible for sspABCD gene cluster,is found in Vibrio cyclitrophicus FF75 and named DNA phosphorothioation modification of type II.It reveal that the strains that can grow at 4? are unable to grow normally at 15? when the modified gene cluster was deleted.It's further found that this phenomenon is caused by the sspE gene downstream of the modified gene.That is to say,sspE gene can inhibit the growth of mutants missing with phosphorothioation modified gene at low temperature.However,the mechanism of the phenomenon and the function of the encoded proteins are still need to be further explored.In this study,we focused on the expression,purification and crystallization of SspE protein in DNA phosphorothioation modification of type II,aiming to obtain the three-dimensional structure of the protein and lay a theoretical foundation for the further study of its physiological activity.The SspE protein of Vibrio FF75 were most in the form of inclusion bodies,which was not conducive to the subsequent purification and crystallization.Therefore,the SspE protein of Streptomyces yokosukanensis DSM 40224 with high homology with V.cyclitrophicus FF75 was replaced.Finally,after optimization and screening,we obtained wild type and selenium SspE protein crystals with X-ray diffraction resolution of 3.3 angstrom.The phase problem was solved by single wavelength anomalous scattering method.However,the resolution did not meet the requirement of analytic structure,we only got the crystal structure of the N-terminal(1-327AA)part in the SspE protein.While the C-terminal part of the protein can only be seen the main sequences with the functional groups of the side chain resolved.The optimization of SspE protein crystal is still in progress.