DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

The replication of eukaryotic genomes follows a temporally staged program, in which late origin firing often occurs within domains of altered chromatin structure(s) and silenced genes. Histone deacetylation functions in gene silencing in some late-replicating regions, prompting an investigation of the role of histone deacetylation in replication timing control in Saccharomyces cerevisiae. Deletion of the histone deacetylase Rpd3 or its interacting partner Sin3 caused early activation of late origins at internal chromosomal loci but did not alter the initiation timing of early origins or a late-firing, telomere-proximal origin. By delaying initiation relative to the earliest origins, Rpd3 enables regulation of late origins by the intra-S replication checkpoint. RPD3 deletion suppresses the slow S phase of clb5⌬ cells by enabling late origins to fire earlier, suggesting that Rpd3 modulates the initiation timing of many origins throughout the genome. Examination of factors such as Ume6 that function together with Rpd3 in transcriptional repression indicates that Rpd3 regulates origin initiation timing independently of its role in transcriptional repression. This supports growing evidence that for much of the S. cerevisiae genome transcription and replication timing are not linked.Duplication of eukaryotic chromosomes is a temporally regulated process that involves accurate replication of DNA sequences coupled with assembly of chromatin. In general, transcriptionally active euchromatin replicates before the structurally condensed and transcriptionally silent heterochromatin. The significance of this epigenetic phenomenon remains to be determined; however, the temporal dynamics of chromosomal replication are thought to be mechanistically linked to the establishment and inheritance of gene expression programs encoded in the chromatin structure. Altered replication timing is associated not only with changes in gene expression, but also with chromosome instabilities and human cancers, suggesting that temporal staging of replication may contribute to the fidelity of genome duplication (reviewed in reference 21).DNA replication initiates at specific sites distributed along each chromosome that are termed origins of replication (reviewed in references 4 and 20). Individual origins exhibit characteristic activities defined by their timing of activation in S phase and their likelihood of activation in each S phase (origin efficiency). The activation characteristics of individual origins, their relative chromosomal locations, and normal impediments to replication fork progression determine the overall dynamics of genome replication. Recent genome-wide studies of origin locations and replication timing have revealed much about the replication dynamics and organization of the Saccharomyces cerevisiae genome (36, 54). Nevertheless, differential origin efficiency and activation timing depends upon a largely undefined relationship between chromatin structure and replication initiation mechanisms.In S. cerevisiae, origin relocation...

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3⌬ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.[Keywords: DNA replication fork; DNA damage; DNA repair; cell cycle checkpoint; BrdU; phosphatase; microarray] Supplemental material is available at http://www.genesdev.org.

Background: Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites.

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5⌬ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5⌬ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5⌬ cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5⌬ cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.Duplication of eukaryotic chromosomes involves the initiation of DNA synthesis from multiple origins of replication distributed along each chromosome. Although chromosomal DNA replication is restricted to the S-phase period of the cell cycle, individual replication origins initiate DNA synthesis (or "fire") at different times during S phase in a regulated fashion such that each replication origin has a characteristic initiation time within S phase. The exact nature of this regulation is not fully understood but appears to be influenced by two apparently unrelated factors: the local chromatin environment of each origin, which establishes the relative order of origin firing prior to S phase (reviewed in reference 43); and checkpoint signaling pathways, which modulate the extent to which the initiation of certain origins is delayed, particularly in response to replication defects or DNA damage (21,29,31,33).The pre-replicative complex (pre-RC) assembles at and governs the function of each replication origin (reviewed in reference 4). Activation of the pre-RC results in its conversion into two, divergent, replication fork complexes (replisomes) through the recruitment of additional DNA synthesis factors, such as Cdc45 and DNA polymerases. Pre-RC activation requires the function of cyclin-dependent kinase (CDK), which consists of a catalytic subunit (Cdk1) controlled by a cyclin subunit whose expression and stability are cell cycle regulated. In Saccharomyces ce...

The membrane guanylate cyclase of vertebrate photoreceptor rod outer segments plays a key role in controlling the levels of cGMP, the intracellular second messenger that mediates visual signaling (1-5). In the dark-adapted photoreceptor, the resting cGMP levels are set by the balance of synthesis and degradation due to the basal activities of the guanylate cyclase and the cGMP phosphodiesterase. Throughout the processes of phototriggering, photorecovery, and light adaptation, the activity of the guanylate cyclase appears to be modulated to control cGMP levels. Several factors are known to regulate the guanylate cyclase activity. Intracellular calcium concentration in the range of 0.1 to 1 M inhibits cyclase activity in the presence of small calcium-binding proteins, GCAPs 1 (6 -8). In contrast, a protein of 40 kDa, composed of a multimer of 6 -7-kDa peptides, activates the cyclase in a calcium-dependent manner with halfmaximal activation occurring between 2 and 5 M (9). In addition, guanylate cyclase activity has been reported to be modulated by ATP (10, 11) and by protein kinase C (12). The calcium regulation mediated by GCAPs is thought to be responsible for cyclase activation during photorecovery and light adaptation; however, the physiological significance of regulation by the other factors is unclear. In all cases, the mechanisms by which these factors control the activity of the photoreceptor guanylate cyclase are not understood.The primary structure of the photoreceptor membrane guanylate cyclase has been deduced from cDNA sequences isolated from human, bovine, and rat retinas (13-15). 2 The information indicates that it is a member of the family of membrane guanylate cyclases that function as receptors for peptide ligands. Four functional domains are characteristic of these proteins: an amino-terminal "extracellular" domain, a transmembrane region, an intracellular protein kinase-like domain, and a carboxylterminal guanylate cyclase catalytic domain (17)(18)(19). In other members of the family, binding of peptide ligands to the extracellular amino-terminal domains induces stimulation of cytoplasmic guanylate cyclase activity. Molecules known to function in this manner include receptors for ANP (GC-A) and CNP (GC-B), guanylin and Escherichia coli enterotoxin (GC-C), and the sea urchin egg peptides, speract and resact. Although no ligand has been identified for the photoreceptor guanylate cyclase, its structural similarity to the receptor guanylate cyclases suggests that it may be regulated in a similar manner.In this work, we have focused on the role of the kinase-like domain of the photoreceptor guanylate cyclase. Studies of other members of the receptor guanylate cyclase family clearly indicate that the kinase homology domain is required to transduce the ligand-binding signals and activate the guanylate cyclases (20 -22). ATP is required for ligand-mediated activation, but nonhydrolyzable ATP analogues work as well (17). No kinase activity has been measured for any receptor guanylate cyclase, and autoph...

No abstract

Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differing maturation states from committed progenitors to differentiating neurons. These observations will facilitate molecular characterization of PSC-derived RGCs during differentiation, critical knowledge for establishing the veracity of these in vitro produced cells. Furthermore, observations made in the retinal organoid model closely parallel those in human fetal retina further validating use of retinal organoid to model early retinal development.