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S phase ( Synthesis phase) is the phase of the cell cycle in which DNA is DNA replication, occurring between G1 phase and G2 phase. Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.
Accordingly, entry into S-phase is controlled by molecular pathways that facilitate a rapid, unidirectional shift in cell state. In yeast, for instance, cell growth induces accumulation of Cln3 cyclin, which complexes with the cyclin dependent kinase CDK2. The Cln3-CDK2 complex promotes transcription of S-phase genes by inactivating the transcriptional repressor Whi5. Since upregulation of S-phase genes drive further suppression of Whi5, this pathway creates a positive feedback loop that fully commits cells to S-phase gene expression.
A remarkably similar regulatory scheme exists in mammalian cells. signals received throughout G1-phase cause gradual accumulation of cyclin D, which complexes with CDK4/6. Active cyclin D-CDK4/6 complex induces release of E2F transcription factor, which in turn initiates expression of S-phase genes. Several E2F target genes promote further release of E2F, creating a positive feedback loop similar to the one found in yeast.
Activation of the pre-RC is a closely regulated and highly sequential process. After Cdc7 and S-phase CDKs phosphorylate their respective substrates, a second set of replicative factors associate with the pre-RC. Stable association encourages MCM helicase to unwind a small stretch of parental DNA into two strands of ssDNA, which in turn recruits replication protein A (RPA), an ssDNA binding protein. RPA recruitment primes the replication fork for loading of replicative DNA DNA polymerase and PCNA sliding clamps. Loading of these factors completes the active replication fork and initiates synthesis of new DNA.
Complete replication fork assembly and activation only occurs on a small subset of replication origins. All eukaryotes possess many more replication origins than strictly needed during one cycle of DNA replication. Redundant origins may increase the flexibility of DNA replication, allowing cells to control the rate of DNA synthesis and respond to replication stress.
In addition to increasing transcription of histone genes, S-phase entry also regulates histone production at the RNA level. Instead of Polyadenylation, canonical histone transcripts possess a conserved 3` Stem-loop motif that selective binds to Stem Loop Binding Protein (SLBP). SLBP binding is required for efficient processing, export, and translation of histone mRNAs, allowing it to function as a highly sensitive biochemical "switch". During S-phase, accumulation of SLBP acts together with NPAT to drastically increase the efficiency of histone production. However, once S-phase ends, both SLBP and bound RNA are rapidly degraded. This immediately halts histone production and prevents a toxic buildup of free histones.
For large genomic regions, inheritance of old H3-H4 nucleosomes is sufficient for accurate re-establishment of chromatin domains. Polycomb Repressive Complex 2 (PRC2) and several other histone-modifying complexes can "copy" modifications present on old histones onto new histones. This process amplifies epigenetic marks and counters the dilutive effect of nucleosome duplication.
However, for small domains approaching the size of individual genes, old nucleosomes are spread too thinly for accurate propagation of histone modifications. In these regions, chromatin structure is probably controlled by incorporation of histone variants during nucleosome reassembly. The close correlation seen between H3.3/H2A.Z and transcriptionally active regions lends support to this proposed mechanism. Unfortunately, a causal relationship has yet to be proven.
In addition to these canonical checkpoints, recent evidence suggests that abnormalities in histone supply and nucleosome assembly can also alter S-phase progression. Depletion of free histones in Drosophila cells dramatically prolongs S-phase and causes permanent arrest in G2-phase. This unique arrest phenotype is not associated with activation of canonical DNA damage pathways, indicating that nucleosome assembly and histone supply may be scrutinized by a novel S-phase checkpoint.
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