TRF2 recruits ORC through TRFH domain dimerization.

Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication.

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27(Kip1) was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF(Skp2) ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF(Skp2) pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.