Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.Agencia Estatal de Investigacion [AEI/10.13039/5011000 ´ 11 033]; Ministerio de Ciencia e Innovacion, and co-´ funded by the European Regional Development Fund(ERDF-UE) [PID2020-114429RB-I00 to O.L., PID2020-112998GB-100 to F.M.-H]; Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund [Y2018/BIO4747 and P2018/NMT4443 to O.L. and F.M.-H.]; National Institute of Health Carlos III to CNIO; J.R.L.O. and O.N. acknowledge support from the Molecular Interactions Facility at the CIB-CSIC; N.G.-R. was supported by a Boehringer Ingelheim Fonds PhD fellowship; N.F.L. is funded by NIH [GM107287]. Funding for open access charge: Agencia Estatal de Investigacion [AEI ´ /10.13039/501100011 033]; Ministerio de Ciencia e Innovacion, co-funded by the Eu-ropean Regional Development Fund (ERDF) [PID2020-114429RB-I00].Peer reviewe

Studying macromolecular interactions of cellular machines by the combined use of analytical ultracentrifugation, light scattering, and fluorescence spectroscopy methods

22 p.-5 fig.Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.This work was supported by the Spanish Ministerio de Ciencia e Innovacion (grant numbers 2023AEP105 and PID2019-104544GB-I00/AEI/10.13039/501100011033 to C.A., B.M., G.R. and S.Z.). M.S.S. was supported by the Agencia Estatal de Investigación and the European Social Fund (grant number PTA2020-018219-I/AEI/10.13039/501100011033).Peer reviewe

Molecular architecture and oligomerization of C. glabrata Cdc13 underpin its telomeric DNA binding and unfolding activity

1 p.-7 fig.The CST complex, composed of Cdc13, Stn1 and Ten1 in yeast, mediates the replication and stability of telomeric DNA. Cdc13, the least evolutionarily conserved component, features four concatenated OB-fold domains, whose architecture and functions remain poorly understood. We dissected the molecular architecture of Candida glabrata Cdc13 and showed how each of its OB folds contributes to its self-association and binding to telomeric DNA sequences. Using a combination of biochemical and biophysical tools, we concluded that all individual domains contribute to DNA binding despite not being directly implicated in the binding itself. Analyzing Cdc13 mutants lacking one or more OB-fold domains, we observed that Cdc13 forms dimers primarily through the interaction between OB-fold 2 (OB2) domains, stimulating the binding of OB3 to telomeric sequences. Furthermore, we showed that C. glabrata Cdc13 and CST form higher-order complexes via oligomerization through OB1. Our results reveal the molecular organization of C. glabrata Cdc13, how this regulates DNA binding, and imply that the distinct architectures of yeast Cdc13 share common principles.Peer reviewe