The Structural Study Of ASAP1-SNI1 Subcomplex Within The SMC5/6 Complex In Arabidopsis

The SMC5/6 is a protein complex which has been identified in various organisms.A plethora of studies have been shown that the SMC5/6 complex is associated with homologous recombination,structural maintenance of ribosomal DNA and heterochromatin,telomere extension and regulation of chromatin topology.The SMC5/6 complex is composed of two SMC proteins,SMC5 and SMC6,and several Nse subunits.Generally,a number of four Nse subunits,namely Nse1-Nse4,have been identified in most organisms.In recent years,two additional subunits,Nse5 and Nse6,have been found in yeast,Arabidopsis and human.Functional studies revealed that Nse5 and Nse6 regulate the localization of SMC5 and SMC6 subunits to the DNA damage site and work together to perform DNA damage repair.In Arabidopsis,the functional counterparts of NSE5 and NSE6 are ASAP1 and SNI1,respectively.Previously,numerous studies have demonstrated that SNI1 function as a negative regulator of systemic acquired resistance,repressing the expression of the defense genes.The dual roles of SNI1 indicate that there might be some connections between plant immune response and DNA damage repair,where SNI1 plays crucial roles in this crosstalk.To elucidate the molecular mechanism,we set out to determine the crystal structure of ASAP1-SNI1 complex.In this study,we conducted a preliminary exploration on the structural study of the ASAP1-SNI1 complex.1)our initial attempts to purify the individual ASAP1 and SNI1 using E.coli expression system proved to be a failure,as both proteins were prone to form inclusion bodies.2)Alternatively,we successfully obtained the ASAP1-SNI1 complex by a co-expression strategy,confirmed the direct interactions of the both proteins.3)We screened approximately 200 truncations of ASAP1 and SNI1 and finally characterized the interaction regions between ASAP1 and SNI1.Though approximately more than 24,000 crystalized conditions have been screened,unfortunately no crystals have been obtained yet.Although the crystal of ASAP1-SNI1 complex was not obtained in this study,we have optimized the expression conditions of ASAP1-SNI1 complex and identified the interaction regions of ASAP1-SNI1 complex,which will provide some clues for the future structural and functional study of ASAP1-SNI1 complex.