This page describes S phase control in fission yeast, and provides links to other information around the net. You can check out a table of known S phase genes.
See this page for a discussion of our lab's research.
The graphic at the right shows you the fission yeast cell cycle, using photographs of DAPI stained pombe. (For more about the pombe life cycle, see the previous page). Although G2 occupies roughly 70% of the cell cycle, the interesting bits to most investigators are DNA replication (S phase), when the chromosomes duplicate, and mitosis (M phase), when the duplicated chromosomes segregate.
Interphase (non-mitotic) cells have a large nucleus with a bite taken out of it; the bite is the nucleolus. Mitotic cells have condensed nuclei which will undergo metaphase, anaphase, and telophase to separate the chromosomes along a simple mitotic spindle. In this image, you can see the metaphase and anaphase cells going from one to two nuclei. The daughter nuclei look as though they bounce off the ends of the cell before coming to rest in their new position.
Cells septate as they enter S phase of the next cell cycle. This explains why pombe has no obvious G1. Our research focuses on the S phase and how it is coupled to other cell cycle events. (Image © S. L. Forsburg; see pictures page for usage info.)
Some of the genes required for the regulation of DNA replication have been identified in fission yeast, and the following figure shows part of the complex regulatory network that has been dissected by a number of different labs.
For more information about particular gene products, click on their names in the figure below (requires that your browser support client side image maps). For gene references, check out our table of S phase genes and gene name converter.
![[a schematic of genetic regulatory pathways]](/~forsburg/images/regulation.gif)
Entry into the cell cycle in fission yeast is rapidly followed by the onset of DNA replication. Below, we provide a brief overview of the events in the diagram.
Entry into the cycle at START
Transition into a cycle of division at START requires the product of the p34cdc2 kinase although the targets and cyclin partner(s) of the kinase at this transition point are unknown. This is roughly equivalent to the G0 to G1 transition in mammalian cells. Transition through START also requires the Cdc10p transcriptional activator. This protein offers one way of coupling START to S phase; forming a complex with Res1p and to a lesser extent, Res2p, Cdc10p activates the transcription of several genes known to be required for DNA replication, including cdc22+, encoding the enzyme ribonucleotide reductase; cdc18+, encoding a replication initiator (CDC6 in other systems), and cdt1+. However, Cdc10 has a phenotype that clearly implicates it upstream of these replication proteins (arrested cells are still proficient for mating), and its START-specific targets are unknown.
Assembly of the pre-replication complex
The earliest stages of S phase involve the assembly and activation of the individual replication origins--there are probably about 400 of them in fission yeast, based on what we know from budding yeast. Data indicate that the pre-replication complex, or preRC, actually forms during M phase by the sequential loading of Cdc18 and Cdt1, and the MCM complex at individual replication origins that are already bound by the Origin Recognition Complex, ORC. (The colors correspond to the animation at the top of the page, and below). The assembled preRC is poised for subsequent activation.
Five members of ORC have been genetically cloned in pombe (the sixth has been identified biochemically): orp1+, orp2+, orp3+, orp4+, and orp5+. All six MCM proteins are known: mcm2+/cdc19+/nda1+, mcm3+, mcm4+/cdc21+, mcm5+/nda4+, mcm6+/mis5+, and mcm7+. You can read more about the MCMs on our research page.
Activation of the preRC: formation of an initiation complex
The trigger that begins replication at individual replication origins requires the activity of two kinases:
p34cdc2 and its S phase cyclin Cig2p, and
Hsk1 kinase and its partner Dfp1. THe exact order of events and cross-talk in these pathways is still unclear. We know that Cdc2 phosphorylates Drc1 protein (nb: the name Drc1 has been used twice in S.pombe; see the gene conversion table for info), which allows it to bind to rad4/cut5+. Mutants in rad4 are not only completely defective for DNA synthesis, but also show serious checkpoint defects; it's a likely Cdc20p (DNA pol-epsilon) associated protein.
For Hsk1, we know that at least Mcm2p(Cdc19p) is a substrate. Hsk1p activity is required for individual origin firing, and may allow remodelling of the MCM complex or binding or activation of additional factors such as Sna41p (CDC45 homologue), RPA, and DNA polymerase alpha (Pol1p) . We assume the cdc23+ gene product acts at this point, based on its homology to the S. cerevisiae gene MCM10/DNA43. Interestingly, MCM proteins appear to become a replication helicase and are required throughout S phase.
It may be convenient to think of the Rad4-Drc1 pathway, and the MCM-Sna41 pathway as twin regulatory mechanisms converging on the origin to allow complete assembly of the enzymes required for DNA synthesis. However, there may well be cross-talk between these two pathways.
None of the known conditional mcm mutants blocks DNA synthesis as well as a rad4 mutant, which suggests that they may be intrinsically leakier, or that you simply don't need very much of them. Instead, the mcm mutants block late in S phase, as do most temperature sensitive cdc18 mutants. In contrast, cells with deletions in cdc18 or cdt1 not only to fail to replicate their DNA, but they fail to prevent mitosis: the cells try to divide even though their DNA is unreplicated. This is called a checkpoint phenotype and indicates that the emergency monitoring system that responds to defects in replication is bypassed or mutated. In the case of these unusual checkpoint-deficient replication mutants, because they never even begin DNA replication, the cell never gets a signal that replication is in progress; thus the intact checkpoint is not activated. Some ORC mutants and rad4/cut5 also share this phenotype.
Regulation of replication: prevention of re-replication
Cdc18p is normally very unstable and is degraded during S phase. This may depend upon the mitotic activity of the p34cdc2 kinase, mediated by the kinase inhibitor Rum1p and the G2 cyclin Cdc13p. Overproduction of Cdc18p can cause cells to repeat DNA replication, without an intervening mitosis. This suggests that Cdc18p is a crucial factor in determining whether or not origins of replication fire.
At this point, you might want to take a look again at the animation--move your mouse over the image below to make it go. (You may need to reload the image. Click on it for an enlarged view.) ORC is green, Cdc18 is purple, and MCMs are
Other proteins
A number of mutants affecting DNA replication after initiation have been found; these mutants are able to complete some synthesis, and arrest late in S phase with the elongated, cdc phenotype. Their ability to arrest depends upon the intact checkpoint system. Most DNA metabolism mutants have this late-S phase arrest phenotype. Some of these mutants correspond to known, conserved genes, such as DNA polymerase delta (cdc6+) or DNA ligase (cdc17+). On the other hand, others are novel genes; for example, the essential cdc24+ gene has no homology to genes in any other species, but genetic analysis suggest that it acts late in S phase along with Dna2p.
Our goal is to understand how these elements are interconnected to allow the coordinated progression of S phase coupled to chromosome segregation, with particular focus on the role of MCM proteins and their kinase Hsk1.
Additional resources for S. pombe DNA replication:
In the absence of cdc22 , a Cdc10 target that encodes ribonucleotide reductase, large subunit, cells arrest prior to DNA replication with a typically elongated cdc phenotype owing to absence of nucleotides. This can be mimicked by treating the cells with the drug hydroxyurea. The cells respond to this by arresting replication, in part by controlling the activity of Hsk1 kinase. This response requires the conserved kinase Cds1, which is not essential for viability unless replication is perturbed. Most replication mutants manage to synthesize quite a bit of DNA but successfully arrest cell cycle progression. This is another way of invoking a replication checkpoint; in this case, a damage specific checkpoint that requires another conserved kinase, Chk1. Both of these checkpoint pathways require the Rad3 kinase. A review of checkpoint-dependent pathways is beyond the scope of our purpose here; try these lecture notes for more information.
Our table of fission yeast S phase genes and references
Our gene conversion table provides the S. cerevisiae equivalent of over 100 fission yeast genes
You can read a more comprehensive online review about yeast DNA replication by Joel Huberman, although it is slightly out of date now.
Visit a giant diagram showing the regulation of the entire pombe cell cycle!
Interested in repair and recombination? Visit this table of rad genes.
After replication comes mitosis. Visit Mitosis World
Go to Forsburg Lab research page!
Background information
This section provides links to other sites with background information about DNA replication, the cell cycle, chromatin structure, genetics and cancer.
from the Nobel Prize website--Play the cell cycle game!
Cellular DNA replication requires precise regulation to ensure first, that the genome is replicated once completely in each cell cycle, and second, that the process of replication is coordinated with proper cell cycle entry and subsequent nuclear division. These regulatory requirements exist in all eukaryotes.
Basic information about the process of DNA replication is found at the following sites (these go out of date frequently, so there are a number of options):
© S. L. Forsburg .
