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.

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA–protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4–XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4–ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.

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Using a rat dextran sulfate sodium (DSS)-colitis model, we elucidated that the expression of miRNAs in colorectal tissues, plasma, and feces, particularly miR-31a-5p, miR-181b-5p, and miR-223-3p, could be used as noninvasive biomarkers to evaluate the reversibility of the model. We further investigated whether changes in miRNA levels were reproducible in chronic DSS-induced colitis in rats. Male SD rats were administered 5% DSS in drinking water for two cycles. Cycle 1 consisted of a 7-d dosing period and 14-d recovery period, followed by Cycle 2 consisting of a 5-d dosing period and 7-d recovery period. In-life parameters and the disease activity index (DAI) were respectively examined or calculated daily. Colon length and pathological changes were assessed postmortem in Cycle 2. A panel of nine miRNAs was also measured in colorectal tissues, plasma, and feces using digital PCR. The changes in DAI score and colon length were evident in Cycle 2. Erosive and inflammatory changes were observed in the colon and rectum following DSS treatment. At the end of the off-dose period of Cycle 2, the histological changes in the rectum worsened, while the colon changes showed recovery. The expression patterns of all miRNAs were almost the same in Cycle 2 when compared to those in a previous study (Kodama et al., 2021). Fecal miR-223-3p could be also a useful non-invasive indicator to evaluate the reversibility in chronic DSS-induced colitis in rats.