Structural And Functional Study Of TERT From Candida Tropicalis And Candida Albicans

Telomerase is a unique reverse transcriptase composed of the catalytic telomerase reverse transcriptase(TERT)and telomerase RNA(TER).Telomerase differs from other reverse transcriptases in its ability to add multiple telomere repeats to its DNA substrate using an RNA template that is part of its TER component,thereby solving the“end replication problem”of linear chromosomes in eukaryotes and maintaining the integrity of the genome.However,the addition of consecutive copies of telomeric repeats involves extremely complex and delicate processes,including the elongation of telomere,exquisitely coordinate the template boundary,dissociate the synthesized RNA/DNA helix,translocation of RNA-DNA duplex and template repositioning for repetitive telomeric addition.Yet the precise molecular mechanism of the telomere DNA synthesis has not been well understood,and the elucidation of the precise molecular mechanism underlying these processes depends largely on high-resolution structures.In addition,species-specific features of the structure and function of telomerase make obtaining a more in-depth comprehension of the molecular mechanism of telomerase particularly important.But,little is known about the structure of telomerase,especially that from yeast is completely unknown.In this study,recombinant telomerase proteins from the Candida species Candida albicans(Ca TERT)and Candida tropicalis(Ct TERT)were highly purified by E.coli expression system.Then the purified Candida TERTs were reconstituted with their core TER in vitro,and the assembled ribonucleoprotein displayed special telomerase activity:telomerases of the Candida species were able to perform one round of processive telomeric addition,but without repeat addition processivity.Furthermore,although the TERTs were assembled with isolated template or linked with the pseudoknot(PK)and/or three-way-junction(TWJ)in cis or in trans,the determined activities were identical to those with TER,indicating that the PK and TWJ were not strictly required for fungal telomerase activity,at least in vitro.Even more surprisingly,Candida TERTs can function as a typical reverse transcriptase in vitro.We explored the structural basis of the observed functional properties by carrying out structural studies of TERT from Candida.Although full-length TERT was not easily crystallized,N-terminal truncated TERT from C.tropicalis in apo(Ct TERT^178-879,Ct TERT^158-745)and complexed with its TWJ(Ct TERT^158-879-TWJ),as well as TERT from C.albicans complexed with its TWJ(Ca TERT^177-867-TWJ,Ca TERT^95-867-TWJ),were crystallized and diffracted to 2.47?,2.84?,2.85?,2.98?and 3.46?respectively.The crystal structure of Ct TERT showed that the C-terminal domain(CTE)of Ct TERT collapsed into the conventional cavity observed in other eukaryotic telomerase,in a"closed"state.Subsequently,we performed crystal structure analysis and SAXS experiments,and found that the"closed"state was not an artifact of crystal stacking,but an intrinsic structural property of telomerase,and was not affected by RNA.Surprisingly,contrary to the"closed"spatial conformation of Ct TERT,the TRBD-RT-CTE of Ca TERT formed a ring-like shape,in an"open"state,and the Ca TERT-TWJ complex was a dimer.Combined with structural analysis and biochemical experiments,we revealed the structural basis of Ca TERT-TWJ to form a dimer.In addition,analyzing the crystal structure of the complex of TERT and TWJ(Ct TERT-TWJ and Ca TERT-TWJ),we revealed the molecular mechanism of TERT recognizing TWJ in yeast.Most importantly,we directly proved and accurately revealed the interaction between P6.1 of TWJ and CTE domain of TERT.Later,SAXS and activity assay showed that the CTE of TERT could rotate flexibly,and the conformational change was an intrinsic property of telomerase.Interestingly,molecular dynamics simulation results showed that the conformational change of CTE could dissociate the RNA-DNA hybrid double strand,which was subsequently verified by functional experiments.When analyzing the crystal structure of Candida TERT,we found that there was a U-loop structure in the N-terminal of TRBD,which was very conservative in telomerase through structural analysis and sequence alignment.We named it U-motif.In-depth research on the structure and function of U-motif,we found that U-motif could regulate the synthesis of telomere DNA.Finally,based on the structural analysis and functional experiments of CTE and U-motif,we proposed a molecular model of telomerase synthesis of telomere DNA.