DNA replication is central to cell proliferation. Studies in the past six decades since the proposal of a semiconservative mode of DNA replication have confirmed the high degree of conservation of the basic machinery of DNA replication from prokaryotes to eukaryotes. However, the need for replication of a substantially longer segment of DNA in coordination with various internal and external signals in eukaryotic cells has led to more complex and versatile regulatory strategies. The replication program in higher eukaryotes is under a dynamic and plastic regulation within a single cell, or within the cell population, or during development. We review here various regulatory mechanisms that control the replication program in eukaryotes and discuss future directions in this dynamic field.

DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early-G1-phase at a point referred to as timing decision point. However, how the genome-wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early-S-phase and changes in long-range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture.

We have determined and compared the promoter, coding, and intronic sequences of the urate oxidase (Uox) gene of various primate species. Although we confirm the previous observation that the inactivation of the gene in the clade of the human and the great apes results from a single CGA to TGA nonsense mutation in exon 2, we find that the inactivation in the gibbon lineage results from an independent nonsense mutation at a different CGA codon in exon 2 or from either one-base deletion in exon 3 or one-base insertion in exon 5, contrary to the previous claim that the cause is a 13-bp deletion in exon 2. We also find that compared with other organisms, the primate functional Uox gene is exceptional in terms of usage of CGA codons which are prone to TGA nonsense mutations. Nevertheless, we demonstrate rather strong selective constraint against nonsynonymous sites of the functional Uox gene and argue that this observation is consistent with the fact that the Uox gene is unique in the genome and evolutionarily conserved not only among animals but also among eukaryotes. Another finding that there are a few substitutions in the cis-acting element or CAAT-box (or both) of primate functional Uox genes may explain the lowered transcriptional activity. We suggest that although the inactivation of the hominoid Uox gene was caused by independent nonsense or frameshift mutations, the gene has taken a two-step deterioration process, first in the promoter and second in the coding region during primate evolution. It is also argued that the high concentration of uric acid in the blood of humans and nonhuman primates has developed molecular coevolution with the xanthine oxidoreductase in purine metabolism. However, it remains to be answered whether loss of Uox activity in hominoids is related to protection from oxidative damage and the prolonged life span.

We found that a group of rubromycins and their analogues, a class of quinone antibiotics that possesses benzofuran and benzodipyran rings to form a spiroketal system, strongly inhibited human telomerase as assessed with a modified telomeric repeat amplification protocol. beta- and gamma-Rubromycins and purpuromycin appeared to be the most potent telomerase inhibitors, with 50% inhibitory concentrations (IC(50)) of about 3 microM, and griseorhodins A and C also showed comparable potencies for the inhibition (IC(50) = 6-12 microM). In contrast, opening of the spiroketal system of beta-rubromycin, giving rise to alpha-rubromycin, substantially decreased its inhibitory potency toward telomerase (IC(50) > 200 microM), indicating the essential role of the spiroketal system in telomerase inhibition. A kinetic study of the inhibition by beta-rubromycin revealed a competitive interaction with respect to the telomerase substrate primer, with a K(i) of 0.74 microM, whereas a mixed type inhibition was observed with respect to the nucleotide substrate. beta-Rubromycin was also potent in inhibiting retroviral reverse transcriptases but had virtually no effect on other DNA/RNA-modifying enzymes including DNA and RNA polymerases, deoxyribonuclease, and topoisomerase. Although beta-rubromycin showed nonspecific cytotoxicities, reducing proliferation of cancer cells (IC(50) approximately 20 microM), we conclude that beta-rubromycin appears to be a lead structure for the development of more potent and selective inhibitors of human telomerase.