TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress.

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

Enhanced resolution of molecular recognition to distinguish structurally similar molecules by different conformational responses of a protein upon ligand binding.

MutT distinguishes substrate 8-oxo-dGTP from dGTP and also 8-oxo-dGMP from dGMP despite small differences of chemical structures between them. In this paper we show by the method of molecular dynamics simulation that the transition between conformational substates of MutT is a key mechanism for a high-resolution molecular recognition of the differences between the very similar chemical compounds. (1) The native state MutT has two conformational substates with similar free energies, each characterized by either open or closed of two loops surrounding the substrate binding active site. Between the two substates, the open substate is more stable in free MutT and in dGMP-MutT complex, and the closed substate is more stable in 8-oxo-dGMP-MutT complex. (2) Conformational fluctuation of the open substate is much larger than that of the closed substate. An estimate of associated entropy difference was found to be consistent with the experimentally found difference of entropy contribution to the binding free energies of the two molecules. (3) A hydrogen bond between H7 atom of 8-oxo-dGMP and the sidechain of Asn119 plays a crucial role for maintaining the closed substate in 8-oxo-dGMP-MutT complex. When this hydrogen bond is absent in the H7-deficient dGMP-MutT complex, the closed substate is no more maintained and transition to the more entropically-favored open substate is induced. (4) Thus, this mechanism of the hydrogen bond controlling the relative stabilities of the drastically different two conformational substates enhances the resolution to recognize the small difference of the chemical structures between the two molecules, dGMP and 8-oxo-dGMP.