In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3Δ cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors.

No abstract

No abstract

Prior to S phase, eukaryotic chromosomes are licensed for initiation of DNA replication, and re-licensing is prohibited after S phase has started until late mitosis, thus ensuring that genomic DNA is duplicated precisely once in each cell cycle. Here, we report that over-expression of Cdt1, an essential licensing protein, induced re-replication in Xenopus egg extracts. Geminin, a metazoan-specific inhibitor of Cdt1, was critical for preventing re-replication induced by Cdt1. Re-replication induced by the addition of recombinant Cdt1 and/or by the depletion of geminin from extracts was enhanced by a proteasome inhibitor, which suppressed the degradation of Cdt1 in the extracts. Furthermore, a nuclear localization sequence identified in Xenopus geminin had a significant role in the suppression of re-replication induced by Cdt1. These results suggest that nuclear accumulation of geminin plays a dominant role in the licensing system of Xenopus eggs.

In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription.

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

Transition-metal-catalyzed asymmetric allylic substitution is a useful reaction in organic synthesis. [1] In the reaction with symmetric C nucleophiles such as dialkyl malonates, good yields and high enantioselectivities can now be obtained with an appropriate combination of a transition metal and a chiral ligand. [2][3][4][5] In contrast to the symmetric C nucleophiles, allylic substitution of 3-substituted allylic alcohols B with unsymmetrical C nucleophiles A is a tough and challenging task, because regio-, diastereo-, and enantioselectivities must be controlled (Scheme 1). In the last few years, research has focused on finding catalysts and chiral ligands that favor the formation of branched chiral products D and E in the allylic substitution of a-amino esters A with B.[ 6, 7] We have already reported Pd-mediated asymmetric allylic alkylation of diphenylimino glycinate 1 with several allylic acetates in the presence of the chiral phase-transfer catalyst (PTC) 6 to give the chiral products C with high enantioselectivity (up to 97 % ee).[6a] In contrast to the palladium catalyst, some transition metals, such as Ir, [3] Mo, [4] and W, [5] promote allylic alkylation at the more highly substituted terminus of the allylic substrate. Trost et al. recently reported that Mocatalyzed asymmetric allylic alkylation with azlactones occurs at the more substituted terminus with high regio-, diastereo-, and enantioselectivity.[8] However, there are no reports concerning the asymmetric synthesis of both diastereomers D and E as major products from the same starting materials and the same chiral ligand. We report here the first enantioselective allylic substitutions of 1 catalyzed by an iridium complex of chiral phosphite 10, and the diastereoselective synthesis of the products 4 and 5 by simply switching the base employed (Scheme 2).Our previous work prompted us to examine PTC 6 as a chiral catalyst in Ir-catalyzed allylic substitutions (Table 1). We first carried out the Ir-catalyzed reaction of 1 and benzoate 2 a in the presence of the chiral PTC 6, 50% KOH, [{IrCl(cod)} 2 ] (cod = cyclooctadiene), and (PhO) 3 P (entry 1). The reaction was complete after 8 h at room temperature and gave the branched products 4 a and 5 a as major products (40 % yield, 4 a:5 a = 75:25) but with low enantioselectivity (46 % ee). We next examined the effect of chiral ligands 7-10[9] in place of chiral PTC 6 on the enantioselectivity. The reaction of 1 with 2 a was carried out in the presence of 50 % KOH (3 equiv), [{IrCl(cod)} 2 ] (10 mol %), and chiral phosphites (20-40 mol %). In all cases, no linear product could be detected. Indeed, the Scheme 1. Transition-metal-mediated asymmetric allylic substitution.Scheme 2. Ir-catalyzed asymmetric allylic substitution of 1 with 2 a, a'. Table 1: Ir-catalyzed asymmetric allylic substitution of 1 and 2 a, a' with chiral PTC 6 or various chiral ligands 7-10. [c]

The origin recognition complex (ORC) binds to replication origins to regulate the cell cycle‐dependent assembly of pre‐replication complexes (pre‐RCs). We have found a novel link between pre‐RC assembly regulation and telomere homeostasis in human cells. Biochemical analyses showed that human ORC binds to TRF2, a telomere sequence‐binding protein that protects telomeres and functions in telomere length homeostasis, via the ORC1 subunit. Immunostaining further revealed that ORC and TRF2 partially co‐localize in nuclei, whereas chromatin immunoprecipitation analyses confirmed that pre‐RCs are assembled at telomeres in a cell cycle‐dependent manner. Over‐expression of TRF2 stimulated ORC and MCM binding to chromatin and RNAi‐directed TRF2 silencing resulted in reduced ORC binding and pre‐RC assembly at telomeres. As expected from previous reports, TRF2 silencing induced telomere elongation. Interestingly, ORC1 silencing by RNAi weakened the TRF2 binding as well as the pre‐RC assembly at telomeres, suggesting that ORC and TRF2 interact with each other to achieve stable binding. Furthermore, ORC1 silencing also resulted in modest telomere elongation. These data suggest that ORC might be involved in telomere homeostasis in human cells.