GEN 6: Replicating the Genome Flashcards
Observe the learning outcomes of this session
Fill in the blanks regarding DNA synthesis
Fill in the blanks for this diagram
How many base pairs are there for every error made by DNA polymerase?
- 1 error every 108 base pairs
What does carefully controlling the S-phase and M-phase ensure?
- chromosomes are copied exactly once per S-phase
- M-phase initiates after S-phase completion
- S-phase is always preceded by M-phase
What if the S-phase and M-phases of the mitotic cell cycle are not correctly controlled?
- these controls are required for genome stability
- failures can lead to over or under replication of chromosomes and aberrant chromosome segregation
- this can lead to aneuploidy and chromosomal rearrangements
- in somatic cells, these failures can compromise cell function or survival, lead to mutations that cause uncontrolled cell proliferation and cancer
What specialised cells or situations would the cell cycle differ from the standard mitotic cycle?
- meiotic cells:
- they undergo two rounds of cell division without the intervening S-phase
- so they can halve their chromosome number
- mega-karyocytes:
- these cells produce platelets needed for blood clotting
- they undergo repeated S-phases with no intervening cell divisions
- they become highly enlarged and polyploid (up to 64 sets of chromosomes), which is a process necessary for platelet production
- senescent cells:
- with appropriate signals, some cells exit the cell cycle during G1 to enter G0
- this could represent a reversible resting phase, an extended or indefinite period of dormancy (senescence) or the beginning of terminal cellular differentiation
Describe the origin of replication in prokaryotes
- in prokaryotes, DNA replication starts at a single position in the circular chromosome, which is called the origin of replication
- unwinding of the chromosome at the origin allows two replication forks to form and move in opposite directions around the chromosome until it is entirely duplicated
How are mammalian cells able to divide around every 20 hours, even though it is estimated to take 200 days?
- mammalian cells initiate DNA replication at multiple origins throughout the genome
- so multiple regions of the genomes can be replicated simultaneously
How long does a typical mammalian cell take to complete S-phase?
- within 8 hours
- although specialised cells can do it in as little as 30 mins
What is the estimate of the number of replication origins in human cells?
- about 50,000 replication origins, which is around 1 per 70kb
Why have mammalian cells evolved elaborate mechanisms to prevent origins from firing more than once per S-phase?
- even if the efficiency was 99.99%, for a mammalian cell with 50,000 origins, there would be 5 failures every cycle
What do all eukaryotes bind to for the assembly of proteins required for DNA synthesis?
- they bind to a complex of proteins that serve as a ‘landing pad’
- this complex is called the origin recognition complex (ORC) composed of 6 protein subunits (ORC1 to ORC6)
Describe how DNA replication originates
- Origin Licensing
- Before DNA synthesis can begin, a pre-replicative complex (preRC) is assembled in G1, a process called origin licensing.
- Two proteins, Cdc6 and Cdt1, are needed to recruit the replicative helicases to the ORC.
- The helicases, each composed of six Mcm proteins, remain inactive at this stage. - Origin Activation
- Origin activation (or ‘firing’), and the ensuing S-phase, requires not only the recruitment of multiple initiation and replication proteins, such as DNA polymerase, but also the phosphorylation of many of these proteins
- The phosphorylation that activates these proteins is the result of Cyclin Dependent Kinase (Cdk) activity.
- Cdk activity also phosphorylates and inactivates ORC, Cdc6 and Cdt1.
- The Mcm helicase must also be activated and this is achieved by a different kinase, Dbf4-dependent kinase (DDK) - Completion of Replication
- S-phase can then proceed to completion and entry into G2 phase
What would happen if origins could be licensed again for replication while S-phase is still in progress?
- it would eventually overwhelm the replication machinery
- there is evidence that DNA re-replication caused impaired cell proliferation, DNA damage, genome instability, cell death or oncogenesis
How does the cell prevent uncontrolled origin firing in the S-phase?
- the inhibitory phosphorylation of ORC, Cdt1and Cdc6 by Cdk
- use of the same kinase activity both to promote origin firing and to inactivate these origin licensing components ensure that each origin fires no more than once per S-phase
- even after S-phase, in G2 and until cells have passed through M-phase and successfully segregated their newly replicated chromosome, origin relicensing must be suppressed to avoid DNA over-replication
- cells have evolved additional mechanisms to prevent unwanted origin licensing, including the binding and inactivation of Cdt1 by geminin, a protein that accumulates in S and G2
Which statement is true about a single replication bubble?
- it must usually fuse with two others before S-phase can be completed
Which statement is true of human origins of DNA replication?
Choose the best answer
- they determine where DNA polymerase will begin DNA synthesis
Which of the following are part of origin licensing?
- the formation of a pre-replication complex
- the loading of Mcm helicases at or near ORC sites
- interactions between Cdt1 and Mcm helicase
Preventing an origin from firing more than once per S-phase requires:
- S-Cdk to inhibit origin licensing.
- DDK to activate the DNA helicase.
- S-Cdk to phosphorylate DNA polymerase.
- S-Cdk to phosphorylate and inactivate Cdt1
- S-Cdk to phosphorylate and activate DNA helicase
- S-Cdk to inhibit origin licensing
- S-Cdk to phosphorylate and inactivate Cdt1
What does Cdk activity regulate apart from promoting entry into S-phase and suppressing origin relicensing?
What are the two Cdk activities called?
- Cdk activity is also the key regulator of entry into M-phase
- these two Cdk activities are sometimes called:
- S-Cdk
- M-Cdk
What are Cdks?
- they are serine-threonine protein kinases
- they transfer a phosphate group from ATP onto certain serine or threonine residues in their protein substrates
- this may activate or inactivate the substrate, depending on the substrate
- they are kinases that must bind a cyclin protein to be active
How are Cdk activities in S and M phase different?
- different substrates are phosphorylated
- this may reflect associations with different cyclins or simply higher kinase activity in M compared to S phase
- M-Cdk substrates are proteins involved in key mitotic events
- such as breakdown of the nuclear envelope and chromosome condensation
- exactly how S-Cdk and M-Cdk phosphorylate different substrates is not yet fully understood and there are two models to help explain
Describe the qualitative model for Cdk activity
- Different cyclins accumulate at specific cell cycle stages:
- S-cyclins at S-phase
- M-cyclins at M-phase
- This suggests one way in which the same Cdk can have different effects at different cell cycle stages: by associating with different cyclins it acquires different substrate specificities at S- and M-phase.
- This makes the action of S-Cdk and M-Cdk qualitatively different and is termed the qualitative model
Describe the quantitative model for Cdk activity
- The quantitative model proposes that S-Cdk and M-Cdk activities differ only quantitatively and that M-phase substrates require higher levels of Cdk activity to become phosphorylated than the S-phase substrates
- As cyclin, and therefore Cdk activity, accumulates, two successive thresholds of Cdk activity are reached, the first (TS) at the G1/S boundary, the second (TM) at the G2/M boundary, to promote S- and M-phases, respectively
Are the qualitative and quantitative models for Cdk activity mutually exclusive?
- they are not mutually exclusive
- it is possible that they are combined in different ways to generate cell cycles suited to different cell types
How is the M-phase inactivated to allow the next S-phase?
- before a new S-phase can begin, the replication origins have to be re-licensed
- this is achieved by proteolysis of the cyclin so that Cdk activity is no longer sufficient to inhibit origin licensing
- cyclins are degraded when a ubiquitin ligase complex (anaphase-promoting complex / cyclosome or APC/C) tags them with ubiquitin, which marks them for degradation by the cell
- during S, G2 and early M, Cdk activity inhibits APC/C
- in late mitosis, however, M-Cdk activates APCC activity which leads to degradation of cyclin but also cohesin and the licensing inhibitor geminin
- so, the same signal that triggers mitotic chromosome segregation also ensures that cells exit mitosis into a G1 state that permits origin relicensing
What makes S-phase dependent on completion of mitosis?
- In summary, high M-Cdk activity, by activation of APC/C, initiates both chromosome segregation and, via cyclin degradation, the loss of its own activity.
- As a result, cells enter G1 only after chromosome segregation and with sufficiently low Cdk activity to allow origin relicensing and the next S-phase
- In this way, S-phase is dependent on completion of mitosis.
What does this diagram show us?
- it tells us about the alternating and counterbalancing phases of Cdk and APC/C during cell cycle
What may DNA damage do to Cdk activity?
- Cdk activity is also inhibited in order to cause cell cycle arrest in response to certain signals
Fill in the blanks
Name a type of protein that acts as an enzyme cofactor and whose concentration varies with cell cycle phase as a result of proteolytic degradation
- cyclin
Name a ubiquitin ligase complex activated during mitosis and responsible for degradation of cyclin, geminin and securin
- anaphase-promoting complex (APC/C)
Name a protein heterodimer that drives cells into mitosis
- M-Cdk
Why are there topological stresses on DNA during replication?
- as Mcm helicase unwinds the double-stranded DNA, tension builds up on the DNA
What are the two types of structures that reduce topologically stress on DNA?
- supercoils: ahead of the replication fork
- pre-catenanes: behind the replication form, sister chromatid intertwines
- caption: The helicase activity of replication machinery unwinds the DNA duplex (A). This creates stress both ahead of (B) and behind (C) the replication fork causing the formation of supercoils and precatenanes, respectively.
How and why do supercoils and precatenanes need to be removed?
- so that replication can be completed and sister chromatids can separate
- cells use enzymes called topoisomerases
What do type I topoisomerases do?
- they cleave and reseal single strands in DNA supercoils
- they reduce supercoiling
What do type II topoisomerases do?
- they cleave both strands in DNA supercoils and in precatenanes
- they can reduce supercoiling and resolve precatenanes
How does Topoisomerase I work?
- Topoisomerase I enzymes cut a single strand of the double-helix, pass the other strand through the cut and reseal the break
- relaxing the overwound molecule which now has one fewer twist
How does Topoisomerase II work?
- topoisomerase II enzymes cut both strands of DNA
- and passes another double-strand through the brak
- if a molecule of DNA is super-coiled then it can remove them two twists at a time, to form a relaxed circle
What happens when Top2 is depleted in human cells?
- sister chromatids remain intertwined at anaphase causing delayed and abnormal chromosome segregation
- causing cell death
What is Top2 used for in medical treatment?
- Drugs that inhibit Top2, by inhibiting its activity after it has cleaved DNA, cause DNA double-strand breaks.
- Such Top2 inhibitors are widely used for cancer chemotherapy.
- It may seem paradoxical that DNA damage, which is known to cause cancer, is also used to treat it.
- This is possible because cancer cells are more sensitive than normal cells to DNA damage.
- Conversely, it is also thought that low concentrations of Top2 inhibitors may be present in foodstuffs and contribute to carcinogenesis
What is the end replication problem?
- the problem arises from the repeated requirement for an RNA primer during synthesis of the lagging strand
- as a result, the 5’ end of the lagging strand is incompletely replicated
- without a mechanism to overcome this, chromosomal telomeres are therefore expected to become progressively shorter with successive S-phases
How is the end-replication problem overcome?
- cells have evolved an enzyme complex called telomerase by synthesising new TTAGGG
- the ends of chromosomes contain a section of G-rich series of repeats, called a telomere
- telomerase recognises the sequence and using an RNA template within the enzyme, it elongates the parental strand in the 5’ to 3’ direction and adds in additional repeats
- the lagging strand is then complete by DNA polymerase-alpha, which carries DNA primase as one of its subunits, so the end is completed copied in new DNA
What are the two main components of telomerase?
- the protein telomerase reverse transcriptase (TERT): 126kDa in size
- a 451 nucleotide non-coding RNA molecule (TERC): acts as a template for TERT to transcribe from
What can TERT and TERC do and what can they not do?
- together they are sufficient to extend TTAGGG repeats in vitro
- but for telomere maintenance in cells, they must interact with other proteins that also bind to telomeres
Look at this diagram and describe what dyskerin and the shelterin complex is
- Dyskerin: is a key telomerase protein, which, along with its three accessory proteins, binds and stabilises TERC
- This ensures TERC is not degraded in the cell and can assemble with TERT.
- The shelterin complex comprises six proteins
- Shelterin helps to protect (or ‘shelter’) telomeric DNA from being recognised as damaged DNA and inappropriately repaired.
Learn the telomere syndromes
Why does the speed at which tissues divide affect the cells?
- tissues that rapidly divide tend to be most affected, as telomeres shorten rapidly without telomerase to repair them, causing cells to senesce or die
What is replicative senescence?
- If cells lack telomerase activity their telomeres shorten with increasing cell divisions until a program of cell senescence is activated.
- This process of replicative senescence is normal for most somatic cells.
- For example, it can be seen when human fibroblasts are grown in culture: after about 50 doublings (known as the Hayflick limit, after its discoverer) they stop dividing and enter a senescent state.
- In most cells, however, replicative senescence is a safeguard against unwanted cell proliferation and oncogenesis.
- Indeed, cancer cells are almost always found to over-express telomerase
Which cells have higher levels of telomerase?
- most cells express low levels of telomerase and are destined to senesce
- higher levels are needed to undergo extensive proliferation:
- germ cells
- early embryo cells
- somatic stem cells
What are some consequences of telomerase disease?
- Telomere length is naturally under very fine control
- Too much expression may cause a predisposition to oncogenesis, while too little may result in premature cellular senescence
- An example of this in telomere disease is premature senescence of haematopoetic stem cells leading to bone marrow failure in patients with dyskeratosis congenita.
What are the effects of down-regulating some telomere proteins?
- Interestingly, although the risk of cancer can be increased by up-regulating telomerase, allowing cells to continue growing, it can also be increased by down-regulating some telomere proteins
- For example, if TRF2 is mutated telomeres are not well protected by shelterin and chromosome ends can fuse with one another or to broken DNA (see the figure below)
- Such events are highly genome-destabilising, mutagenic and therefore potentially oncogenic.
Fill in the blanks
Concerning Topoisomerase II, which is correct
- it is essential for cell viability
- cuts both strands of the DNA double helix
- it is a target for anti-cancer drugs
Select all correct statements
- excessive telomerase activity increases the risk of oncogenesis.
- Impaired telomere maintenance increases the risk of oncogenesis
- Inhibiting telomerase in cancer cells is an acceptable approach in anti-cancer research.
- Many telomere syndrome symptoms can be explained by loss of somatic stem cells.
