▪ Abstract The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.

DNA replication in eukaryotic cells

https://www.cell.com/cell/pdf/S0092-8674(04)00805-0.pdf

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels.

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis

Osmotic homeostasis is one of the most aggressively defended physiological parameters in vertebrates. However, the molecular mechanisms underlying osmotic regulation are poorly understood. The transient receptor potential channel, vanilloid subfamily (TRPV4), is an osmotically activated ion channel that is expressed in circumventricular organs in the mammalian CNS, which is an important site of osmotic sensing. We have generated trpv4-null mice and observed abnormalities of their osmotic regulation. trpv4-/- mice drank less water and became more hyperosmolar than did wild-type littermates, a finding that was seen with and without administration of hypertonic saline. In addition, plasma levels of antidiuretic hormone were significantly lower in trpv4-/- mice than in wild-type littermates after a hyperosmotic challenge. Continuous s.c. infusion of the antidiuretic hormone analogue, dDAVP, resulted in systemic hypotonicity in trpv4-/- mice, despite the fact that their renal water reabsorption capacity was normal. Thus, the response to both hyper- and hypoosmolar stimuli is impaired in trpv4-/- mice. After a hyperosmolar challenge, there was markedly reduced expression of c-FOS in the circumventricular organ, the organum vasculosum of the lamina terminalis, of trpv4-/- mice compared with wild-type mice. This finding suggests that there is an impairment of osmotic sensing in the CNS of trpv4-/- mice. These data indicate that TRPV4 is necessary for the normal response to changes in osmotic pressure and functions as an osmotic sensor in the CNS.

https://www.pnas.org/content/pnas/100/23/13698.full.pdf

Abnormal osmotic regulation in trpv4-/- mice

In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.

https://science.sciencemag.org/content/sci/290/5500/2309.full.pdf

Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.

Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins/epithelial sodium channel (DEG/ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1–0.5%), in the hot-plate test (52.5 and 55 vs. 50°C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate- to high-intensity pain sensation.

https://www.pnas.org/content/pnas/99/13/8992.full.pdf

A role for ASIC3 in the modulation of high-intensity pain stimuli

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G1 phase, and reappeared as cells progressed through G1 phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G1 phase, but became stably bound during mid-G1 phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed.

/pdf/selective-instability-of-orc1-protein-accounts-for-the-484dn15dk0.pdf

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M–G1 transition in mammals

Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors

Serotonin [5-hydroxytryptamine (5-HT)] released from mast cells or platelets in peripheral tissues is one of the important inflammatory mediators in pain and hyperalgesia. The involvement of 5-HT in pain is complex because it could inhibit or facilitate nociceptive transmission, reflecting the presence of multiple 5-HT subtype receptors on peripheral and central nociceptors. The present study aimed to investigate the involvement of 5-HT2B in 5-HT-induced pain and whether the subtype exists in dorsal root ganglion (DRG) neurons. Injecting the 5-HT or 5-HT2 agonist in hindpaws of mice induced significant hyperalgesia to mechanical stimuli, which was inhibited by the 5-HT2B/2C antagonist but not by 5-HT1A, 5-HT2A, or 5-HT3A antagonists. Therefore, 5-HT2B or 5-HT2C may be involved in 5-HT-induced mechanical hyperalgesia. The 5-HT2B/2C antagonist also blocked 5-HT-induced transient [Ca2+] signaling in DRG neurons. All subtypes of 5-HT receptors except 5-HT2C and 5-HT6 are present in DRGs. In situ hybridization also demonstrated 5-HT2B mainly expressed in small- to medium-diameter DRG neurons that respond to pain. Likely, 5-HT2B mediates 5-HT-induced mechanical hyperalgesia in mice.

https://www.jneurosci.org/content/jneuro/31/4/1410.full.pdf

Serotonin Receptor 5-HT2B Mediates Serotonin-Induced Mechanical Hyperalgesia

Background
Tissue acidosis is effective in causing chronic muscle pain. However, how muscle nociceptors contribute to the transition from acute to chronic pain is largely unknown.

https://journals.sagepub.com/doi/pdf/10.1186/1744-8069-10-40

Wei Hsin Sun

Papers

A role for ASIC3 in the modulation of high-intensity pain stimuli

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M–G1 transition in mammals

Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors

Serotonin Receptor 5-HT2B Mediates Serotonin-Induced Mechanical Hyperalgesia

Roles of ASIC3, TRPV1, and NaV1.8 in the transition from acute to chronic pain in a mouse model of fibromyalgia.