The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins.

Genetic instability, a hallmark of cancer, can occur when the replication machinery encounters a barrier. The intra-S-phase checkpoint maintains stalled replication forks in a replication-competent configuration by phosphorylating replisome components and DNA repair proteins to prevent forks from catastrophically collapsing. Here, we report a novel function of the core Schizosaccharomyces pombe checkpoint sensor kinase, Rad3 (an ATR orthologue), that is independent of Chk1 and Cds1 (a CHK2 orthologue); Rad3(ATR) regulates the association of recombination factors with collapsed forks, thus limiting their genetic instability. We further reveal antagonistic roles for Rad3(ATR) and the 9-1-1 clamp - Rad3(ATR) restrains MRN- and Exo1-dependent resection, whereas the 9-1-1 complex promotes Exo1 activity. Interestingly, the MRN complex, but not its nuclease activity, promotes resection and the subsequent association of recombination factors at collapsed forks. The biological significance of this regulation is revealed by the observation that Rad3(ATR) prevents Exo1-dependent genome instability upstream of a collapsed fork without affecting the efficiency of recombination-mediated replication restart. We propose that the interplay between Rad3(ATR) and the 9-1-1 clamp functions to fine-tune the balance between the need for the recovery of replication through recombination and the risk of increased genome instability.

24-hydroxycholesterol replacement rate measured in blood is a non-invasive biomarker of brain demyelination and remyelination in cuprizone-treated mice.

In mice, dietary cuprizone causes brain demyelination with subsequent spontaneous remyelination upon return to normal chow. Heavy water (2H2O) labeling with mass spectrometric analysis can be used to measure brain de novo synthesis of several myelin components including cholesterol, phospholipids, galactocereboside (GalC) and myelin-associated proteins. 24-hydroxycholesterol (24-OHC), the major metabolite of brain cholesterol, is detected in blood and is believed to be specifically derived from CNS cholesterol metabolism. We assessed changes in syntheses of myelin components in brain and of blood sterols during cuprizone-induced experimental demyelination and remyelination, with and without thyroid hormone (T3) treatment. Mice were fed cuprizone for 4 weeks, then returned to control diet and treated with either placebo or T3 (0.005 mg/day). 2H2O was administered for the last 14 days of cuprizone diet, and for either 6, 12 or 19 days of treatment during recovery from cuprizone, after which blood and corpus callosum (CC) samples were collected (n = 5/time point/treatment). 2H incorporation into cholesterol and 24-OHC in blood and CC, and incorporation into phospholipid (PL)-palmitate, GalC, myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) in CC were measured. Cuprizone significantly (p < 0.05) decreased syntheses of cholesterol, 24-OHC, GalC, MBP, CNPase and PL-palmitate in the CC and these effects were all reversed during recovery. T3 treatment significantly (p < 0.05) increased syntheses of cholesterol, 24-OHC and palmitate compared to placebo. 24-OHC and cholesterol turnover rates in brain and blood were nearly identical and 24-OHC rates in blood paralleled rates in CC, indicating that blood 24-OHC derives primarily from the brain and reflects oligodendrocyte function. In summary, changes in synthesis of several lipid and protein components in brain during cuprizone-induced demyelination and remyelination are measurable through stable isotope labeling. Blood 24-OHC turnover rates closely reflect flux rates of brain cholesterol in response to cuprizone and T3, which alter oligodendrocyte function. Labeling of blood 24-OHC has potential as a non-invasive marker of brain de novo cholesterol synthesis and breakdown rates in demyelinating conditions.

Replication fork arrest, recombination and the maintenance of ribosomal DNA stability.

There is increasing interest in the role of replication fork arrest and collapse in stimulating genomic instability. Changes in the copy number of the rDNA repeats mediated by homologous recombination has been linked to programmed replication fork barriers (RFBs) and this has been proposed to serve as a paradigm to help understand the links between replication and recombination. Here we review recent advances in our understanding of the initiation and regulation rDNA recombination and discuss historical observations in the context of recently developed models. We contrast the outcome of replication fork arrest at the rDNA RFB with those at an alternative RFB and suggest that, while there are potential similarities in response, there are also important differences which reflect the highly specialised nature of rDNA metabolism.

Measurement of pancreatic islet cell proliferation by heavy water labeling.

We describe a sensitive technique for measuring long-term islet cell proliferation rates in vivo in rats. Pancreatic islets were isolated and the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of DNA was measured by GC-MS. The results of heavy water labeling and BrdU staining were compared. The two methods were highly correlated (r = 0.9581, P < 0.001). Based on long-term heavy water labeling, approximately 50% of islet cells divided in rats between 8 and 15 wk of age. Of interest, long-term BrdU administration suppressed proliferation of islet cells significantly, but not of bone marrow cells. Physiological evidence further supported the validity of the method: older animals (24 wk old) had 60% lower islet cell proliferation rates than younger rats (5 wk old), and partial (50%) pancreatectomy increased proliferation by 20%. In addition, cholecystokinin-8 treatment significantly stimulated proliferation in pancreatectomized rats only. In summary, heavy water labeling is a quantitative approach for measuring islet cell proliferation and testing therapeutic agents.