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Year 3: Genes and Gene Expression in Eukaryotic Cells > DNA replication and the cell cycle > Flashcards

DNA replication and the cell cycle Flashcards

1
Q

What processes occur during S phase?

A
  • Unfold chromatin structure
  • Copy (replicate) DNA completely and accurately
  • Refold DNA back into chromatin
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2
Q

What are the stages of DNA replication?

A
  • Initiation
  • Elongation
  • Termination
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3
Q

Why is DNA replicated in a semi-discontonuous fashion?

A
  • Because one of the strands is replicated in a continous fashion - strand produced is made using one primer and so is made as one long strand
  • The other strand is replicated in a discontinous fashion - strand produced needs multiple primers and so is made up of lots of small okazaki frgaments
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4
Q

Why is DNA replication bi-directional?

A
  • Because the 2 replication forks produced move in opposite directions from the origin of replication at the same time
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5
Q

Which proteins are active at the replication fork during DNA replication?

A
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6
Q

Describe some characteristics of Helicase

A
  • Helicase is a complex of 6 MCM proteins (MCM2-MCM7) complexed with cdc45 and GINS complex
  • MCM = Minichromosome maintainence
  • 6 MCM proteins + cdc45 + GINS complex = CMG helicase
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7
Q

As well as CMG helicase there are site specific helicases that unwind DNA a t specific regions of DNA. Give some examples of these helicases

A
  • WS helicase - Involved in repication of ribosomal repeats
  • PIF helicase - Involved in replication of telomeres
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8
Q

Which proteins are active at the leading strand during DNA replication?

A
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9
Q

What are the functons of the proteins that are active at the elading strnad during DNA replication?

A
  • Polymerase (Epsilon/delta) - Synthesises DNA
  • Rpa (replication protein a) - binds to single stranded DNA to prevent it from forming secondary structures (has other functions)
  • Pcna - processivity factor for polymerase
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10
Q

How does Pcna increase processivity of polymerase?

A
  • 3 Pcna’s join to form a ring-like structure
  • Structure then binds around the DNA and then binds the polymerase
  • The Pcna structure allows for the polymerase to re-bind to the DNA very quickly whenever it dissociates thus increasing its processivity
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11
Q

What proteins are active at the lagging strnad during DNA replication?

A
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12
Q

Replication of each okazaki frgament is like a miniture version of the whole process of DNA replication and so has stages like inititation etc. What proteins are involved in okazaki frgament initiation and what are their functions?

A
  • DNA polymerase alpha
    • Polymerase - Synthesises DNA
    • Primase - Synthesises RNA primer
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13
Q

Describe the structure of DNA polymerase alpha

A
  • Contains 4 subunit
    • 180 subunit (polymerase activity)
    • 73 subunit
    • 50 and 60 subunit (primase activity)
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14
Q

Describe the initial steps that occur in Okazaki fragment replication including “polymerase switching”

A
  1. Polymerase alpha binds to the lagging strand and synthesises a 10-12bp RNA primer
  2. Polymerase alpha then switches and begins to synthesie DNA complementary to the lagging strand from the RNA primer
  3. After 20-40 bp’s of DNA is synthesised polymerase alpha stops and polymerase epsilon/delta take over the DNA synthesis (this process is called polymerase switching)
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15
Q

Why does polymerase alpha only synthesise 20-40bp of DNA during okazaki frgament replication?

A
  • Because unlike polymerase epsilon/delta polymerase alpha doesn’t have active proof-reading activity
  • This means if polymerase alpha synthesised an entire DNA strand there would be a lot of errors within it
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16
Q

Describe the process of polymerase switching

A
  1. Polymerase alpha interacts with Rpa
  2. This interactions causes polymerase alpha to unbind the DNA
  3. rfc (replication factor c) binds to the template DNA and the primer associated with it which allows for pcna to bind
  4. Once pcna binds it allows for DNA polymerase epsilon/delta to bind which allowd for the synthesis of the DNA to continue
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17
Q

Explain how the okazaki fragments are joined together

A
  1. The RNA primer and about 1/2 of the DNA synthesised by DNA polymerase alpha are removed by Rnaseh, Fen1 or DNA2
  2. Then Rfc, Pcna and polymerase delta bind to the DNA synthesised by DNA polymera delta and synthesise more so that the gap between the 2 okazaki fragments is really small
  3. The really small gap between the 2 okazaki fragments is joined by DNA ligase 1
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18
Q

How was the process and all the proteins involved in elongation discovered?

A
  • Genetics (yeast mutations) e.g. cdc and mcm
  • Biochemistry (SV40 in vitro replication system) - Uses host factors for its replication apart from initiation phase
    • This meant proteins involved in DNA replication could be extracted from SV40-infected cells that could replicate SV40
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19
Q

What are the 2 main components involved in initiation of DNA replication?

A
  • DNA component - origin of replication
  • Protein components - machinery of initiation
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20
Q

What does the origin of replication look like in yeast?

A
  • The origin of replication in yeast is called ARS (Autonomously replicating sequence)
  • It is made up of the following:
    • A domain - 11 bp AT rich region which is required
    • B domaIns (B1, B2, B3) - Not required, Increase efficiency of origin, variable between different origins
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21
Q

What does the origin of replication look like in higher eukaryotes?

A
  • Structure of origins of replications haven’t been discovered yet
  • Likey to be long structured region
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22
Q

What are the main proteins involved in initiation?

A
  • Orc (Origin recognition complex) - Made up of Orc 1-6
  • Cdc6
  • Cdt1
  • MCM complex
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23
Q

Describe the stages of the initiation pathway

A
  1. Orc binds to DNA
  2. This causes both cdc6 and cdt1 to bind to the Orc
  3. Cdt1 the mediates the binding of the MCM complex to form the pre-replication complex (PreRC)
  4. Cdt1, Cdc6 and Orc all get phosphorylated
  5. In response Cdt1, Cdc6 and Orc may all dissociate from the DNA
  6. Then proteins involved in elongation such as cdc45, rpa, DNA polymerase epsilon and DNA polymerase alpha all bind to the DNA - binding is mediated via the pre-initiation complex
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24
Q

How was the initiation pathway discovered?

A
  • Yeast genetics (s.cerevisiae and s pombe)
  • Frog (xenopus) in vitro replication system - Frog oocytes have all the material needed fro DNA replication and so were used to replicate specific pieces of DNA
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25
Q

What are replication fork barriers?

A
  • Termination sites that have been seen by 2D gel analysis as sites where the replication forks stop
26
Q

Where on the DNA are replication fork barriers usually located?

A
  • Usually located in regions of DNA where both replication and transcription need to occur at the same time
  • E.g. Histone genes
27
Q

What structure is produced when a replication fork and a transcription fork collide with each other?

A
  • R-loop - structure containing both DNA and RNA
28
Q

There are thousands for origins of replication within a eukaryotic nucleus. Does initiation of replication occur at everyone of these origins at the same time?

A
  • No, not all origins fire at the same time
  • Genome is divided into early and late replicating regions so origins of replication in those regions will fire at different times
  • Early replicating associated with transcribing euchromatin
  • Late replicating associated with silent (mostly) and highly structured heterochromatin
29
Q

What are the early and late replicating regions of the genomes associatred with?

A
  • Early replicating associated with replicating euchromatin
  • Late replicating associated with replicating highly structured heterochromatin
30
Q

Explain the concept of repliaction factories

A
  • Replication proteins are not just floating free in the nucleus but seem to be held in massive complexes at sepcific points in the nucleus called replication factories - around 300 of them
  • There are multiple replication forks within each factory
  • There are both early and late replicating factories
  • DNA is wound through these replication factories
31
Q

Explain the medical significance of some of the proteins involved in replication

A
  • Pcna/mcm/cdc6 - basis for screening kits
    • These factors are only present in cells that are replicating and appear in these cells before they replicate so can be used for early diagnosis of some diseases
  • Lagging strand factors e.g. FEN - may be involved in trinucleotide repeat expansion
  • WS helicase - Mutated in Werners syndrome
32
Q

What proteins are involved in chromosome condenstaion during prophase?

A
  • Condensin (mainly)
  • Topoisomerase
33
Q

How is condensin involved in chromosome condenstaion?

A
  • Condensin proteins wrap around the base of loops of chromosomes
  • The chromosomes are slowly fed through the condensin proteins forming larger loops as the condensin proteins constrict the base of the loops
34
Q

Chromosome congression occurs during metaphase. What is chromosome congression?

A
  • The process by which chromosomes align at the equator of the cell
35
Q

What structures are involved in chromosome congression?

A
  • Kinetochore
  • Mitotic spindle
  • Spindle pole bodies
36
Q

Describe the structure of the kinetochore

A
  • Divided into 3 main regions - inner, middle and outer
  • DNA - Centromeric heterochromatin (very repetitive)
  • Many different types of proteins within each region e.g.
    • Structural proteins
    • Sequence specific binding proteins
    • Molecular motor proteins
37
Q

Describe the structure of the spindle pole body

A
  • Contain two centrioles orientated at right angles to each other
  • These are surronded by the pericentriolar material (PCM) - associated with many different proteins e.g. gamma tubulin
  • The microtubules protrude outwards from the PCM
38
Q

Describe the structure of the mitotic spindle (microtubules)

A
  • Made up of α and β tubulin which are organised to form a helical structure
  • It has a (+) end: where tubulin is added
  • Also has a (-) end: where tubulin is taken away
39
Q

Describe the process of chromosome congression

A
  1. Duplication of the spindle pole body
  2. The newly formed daugher spindle pole body moves to the far end of the nucleus
  3. The nuclear membrane then breaks down
  4. Then from both spindle pole bodies you get the formation of microtubules that contact the centromeres of the chromosomes as well as the astral microtubules
    • The astral microtubules help anchor the spindle pole bodies into the cytoplasm
  5. When a chromosome is attached to a microtubule from both mothr and daughter spindle pole bodies they will line up at the metaphase plate
  6. There’s a dephosphorylation event that signals the end of metaphase and the beginning of anaphase
40
Q

Explain what happens during anaphase

A
  • Anapahse is split into 2 parts
  • Anaphase A: Chromosomes are pulled towards the spindle pole bodies by the combined action of molecular motors and microtubule shortening
  • Anaphase B: Spindle pole bodies move away from each other
41
Q

Explain what happens during telophase

A
  • Chromosomes decondense and the nuclear membrane re-forms resulting in formation of 2 new nuclei
  • Cytokynesis then occurs to separate the nuclei and form 2 daughter cells
42
Q

What systems contributed to our understanding of chromosome condensation?

A
  • Biochemistry and genetics in yeast and biochemistry on frog (xenopus) in vitro extracts
43
Q

What systems contributed to our understanding of kinetochore structure?

A
  • Biochemestry on mammalian cells, biochemistry and genetics on yeast
44
Q

What systems contributed to our understanding of spindle pole bodies and spindle dynamics?

A
  • Biochemistry on human cells and xenopus in vitro extracts
  • Manipulation and microscopy of cells
  • Purification of proteins from many different organsims
45
Q

What is the main form of regulation in the cell cycle?

A
  • Phosphorylation by cyclin-cdk complexes
46
Q

Describe the structure of a cyclin-cdk complex

A
  • Contains Cdk (cyclin-dependent kinase) which is the component that carries out the phosphorylation activity
  • Contains Cyclin which controls the activity of the Cdk and may control the substrates that the Cdk interacts with
47
Q

Give some examples of types of proteins phosphorylated by cdks

A
  • DNA replication initiation proteins
  • Chromosome condensation proteins
  • Proteins involved in mitosis
48
Q

Can proteins be phosphorylated by multiple cdks?

A
  • Yes some proteins can be acted on by multiple cdks with opposite effects
49
Q

Explain what is mean when it is said that the cell cycle is “charcetrised by waves of cdk activity”

A
  • It means that at any point during the cell cycle the processes that are occuring are a result of the activity of all the cdks active at that point
  • E.g. Just before S phase cdk 4 will become active and phosphorylate its target genes allowing the cell to transition into S phase.
    • It’ll also cause the expression of the next cdk needed for S phase
    • The activity of this cdk will increase as a result this cdk will decrease the activity of cdk 4 resulting in “waves’ of cdk activity
50
Q

How can cdk activity be regulated?

A
  • Changes to cyclins
    • Transcription
    • Proteolysis
  • Changes to cdk/cyclin complexes
    • Chemical modification
    • Inhibitors
    • Cell localisation
51
Q

Explain the transcriptional control of the cyclin genes

A
52
Q

Explain how proteolysis on cyclin subunits is carried out

A
  1. A long ubiquitin chain is added the the cyclin subunit e.g. cyclin B by the APC (ubiquitylation)
  2. This targets the cyclin subunit to the proteosome
  3. The proteosome breaks down the cyclin subunit and ubiquitin is released as a result
  • APC = Anaphase-promoting complex
53
Q

Explain how cdk/cyclin complexes can be chemically modified

A
  • Cdc2 (cdk1) can be inhibited when phosphorylated with Wee1 kinase
  • It can also be re-activated when de-phosphorylated by Cdc25 phosphatase
  • Activation of cdc2 results in transition from G2 to M phase
54
Q

Give some examples of Cdk inhibitors and the Cdks they inhibit

A
  • Yeast Cdk inhibitors
    • Sic (sc)
    • Rum (sp)
  • Higher eukaryotic Cdk inhibitors
    • P21 family - cdk 1/2
    • P28 family - cdk 1/2 and cdk 4/6
    • P16 fmaily - cdk 4/6
55
Q

Give an example of cdk/cyclin localisation within the cell cycle

A
  • During interphase cyclin B is in the cytoplasm while its substrates are in the nculeus
  • Then during prophase it is transported into the nucleus allowing it to act on its substrates causing the cell to move into metaphase
56
Q

What things does the cell check for during each of the cellcycle checkpoints?

A
  • G2 checkpoint: Cell checks for incomplete replication and spindle problems
  • G1 checkpoint: DNA damage
  • S phase checkpoint: DNA damage and stalled replication
57
Q

What are some variations of the cell cycle?

A
  • Meiosis
  • Polymitosis
  • Polyploidisation
  • Amplification
58
Q

Explain how meiosis is a variation of the cell cycle

A
  • Meiosis - 2 round sof mitosis with no DNA replication in between resulting in production of 4 gametes
59
Q

What is polymitosis?

A
  • Occurs when the cell carries out multiple rounds of mitosis without any DNA replication in between
  • Occurs in cells which are polyploid (contain more than 2 sets of homologous chromosomes) to reduce chromosome number
60
Q

What is polyploidisation?

A
  • Occurs when a cell goes through multiple rounds of S phase without going through mitosis
  • 2 ways this can occur:
    • Polyploidy - Multiple rounds of DNA replication result in multiple sets of chromosomes being produced which stay separate from each other
    • Polytenisation - After chromosomes are replicated they stay parallel to each other to generate giant chromosomes
61
Q

How is amplification thought to happen?

A
  • Thought to take place via the onion skin model
  • You get multiple rounds of initiation that generate multiple copies of the region of the chromosome being amplified
  • These copies cause that region to “bulge out” but then recombine via homologous recombination and extend the length of the original chromosome

Year 3: Genes and Gene Expression in Eukaryotic Cells (4 decks)

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