Lecture 3: S phase to G2 Flashcards
What happens overall in S phase?
During S phase, DNA is being synthesised.
• There is a switch from cyclin E to cyclin A.
• Cyclin A drives the transcription of mitotic regulators.
• Cyclin B is also transcribed but it is inhibited by wee1 (induced by cyclin A).
• G2M genes were derepressed in G1/S phase by phosphorylation of p107 and p130. They were initially not transcribed, but they can be when Bymb and foxM1 bind.
• Cyclin A is spatially segregated from cyclin B regulators, it is less accessible to regulation by wee1, myt1 and cdc25.
How does DNA replication occur?
DNA replication is one of the most vital cellular processes. It must be performed well all the cell will die or have major issues.
• Both chromatids should be copied once using a template.
• Replication must be complete.
• The two sister chromatids are separated in anaphase.
How does the pre-RC form?
The pre-IC is a protein complex that forms during the initiation step of DNA replication.
• Licensing and firing are two separate processes. Licensing can only occur in a narrow G1/S window.
• Assembly occurs during late M phase and early G1 phase when Cdk activity is low.
• Before this the ORC is by itself. CDC6 is prevented from binding by Cdk1-B. It is degraded by APC/C-CDC20 (late M/G1). Western blots show geminin and cycB are destroyed by APC/C-CDC20 at the onset of anaphase.
• CDT1 is bound to geminin. This is degraded by APC/C-CDC20 during late M phase/G1.
• CDT1 can bind to the ORC, brining CDC6 and MCM2-7 (helicase) with it.
• CDT1 is degraded during S phase to ensure loading doesn’t happen again.
• Cdk2-E/A then allow the loading of polymerase epsilon, this is the replicative polymerase. GINS, CDC45 and MCM10-11 are the firing factors which are loaded as well. GINS and CDC45 stabilise MCM2-7 and allow it to act as a helicase. MCM10-11 are RPA chaperones (prevent single strands creating secondary structures while the helix unwinding).
• Primase creates the first 10 RNA base pairs. Pol α synthesises the next 20 DNA nucleotides. This allows DNA replication to begin.
• CDT1 and CDC6 leave. CDC6 is destroyed by APC-CDH1 in G1. CDT1 is broken down by SCF. CDC6 and aurora A are shown by fluorescence microscopy in control and siCDH1 to be degraded in G1 by APC/C-CDH1.
How does the cell ensure that DNA is only replicated once?
CDT1 must be removed in late S phase to ensure that the preRC cannot assemble again. This is mediated by 2 SCF dependent pathways.
• PCNA binds to CDT1 and Cd2-A phosphorylate it. This leads to CDT1 being ubiquitinated by SCF. It is then degraded by the 26S proteasome.
• Geminin reaccumulates in G2 and binds any newly synthesised CDT1. It inhibits any new pre-RCs forming.
• The origin can’t be relicensed until the next cell cycle when geminin is destroyed.
What is the role of PLK4?
PLK4 is involved in initiating centriole duplication in S phase.
• Overexpression of PLK4 results in multiple centrosomes.
• Knockdown results in a loss of centrosomes.
• PLK4 expression is promoted by CycE/A.
• It is maintained at low levels due to destruction by SCF-BTRCP proteasome activity.
How is centriole maturation and separation controlled in G2/M?
There are two proteins which are mainly involved.
• Nek2 phosphorylates centrosomal proteins such as CNAPI and causes them to dissociate.
• Cdk1-B phosphorylates KIF11. This promotes centrosome separation. KIF11 is a motor protein that moves microtubules along tracks.
What is the role of PLK1?
PLK1 is another polo-like kinase. Mitotic centrioles/centrosomes are relicensed as the cells exit from mitosis. Plk1 is licences centriole maturation and duplication in mitotic exit for form the centrosome.
• The procentriole and parent centriole are distanced.
• Cartwheel is disassembled. This involves STIL and SAS6 being ubiquitinated by APC/C-CDC20. This follows Cdk1-B phosphorylation.
• It is also involved in centriole to centrosome conversion by being recruited to the PCM and activated as Cdk1 phosphorylates it.
