L19 Flashcards
only some Some cells in adult humans are actively proliferating give examples for those
intestinal epithelial stem cells
lymphocytes during immune responses
what type of cells are neurons in terms of proliferation
terminally differentiated
How do mitogens drive progression into S phase?
Mitogen binds to cell surface receptor tyrosine kinase
Ras-Raf-MAPK kinase signalling pathway is triggered
“Immediate early” genes including Myc are expressed
Myc is a transcription factor which upregulates genes including Cyclin D
Cyclin D, together with Cdk4 or 6, forms G1-Cdk
How does G1-Cdk drive progression into S?
A key target of G1-Cdk is Retinoblastoma protein, Rb
In an early G1 cell, Rb binds to and inactivates the transcription factor E2F
Phosphorylation of Rb by G1-Cdk inactivates Rb
E2F is now free to upregulate expression of genes including Cyclin E and Cyclin A
Cyclins E and A associate with Cdk2 to form G1/S-Cdk and S-Cdk
what can prevent G1-Cdk driven progression into S
p53 is a central regulator of checkpoint responses to stress (Lecture 17)
p53 is normally maintained at low levels in cells by Mdm2-mediated degradation
DNA damage activates kinase signalling through ATM/ATR then Chk1/Chk2
Phosphorylation of p53 displaces Mdm2 and p53 is stabilised
p53 acts as a transcription factor to turn on expression of CKIs such as p21 which inhibits G1/S-Cdk activity
what is the main task of S-phase
replication of genomic DNA
Duplication of the centrosomes
What are the major concerns when we think about replicating DNA?
produce exactly one additional copy of each chromosome
be high fidelity
(almost mutation free; 1 nucleotide change per 10^10 nucleotides per cell division)
what are the features of semi conservative replication in prokaryotes
Requires an origin of replication: initiation
Involves replication forks: elongation
describe initiation of bacterial DNA replication
Circular bacterial chromosomes have a single origin of replication defined by DNA sequence
Specific proteins form an Origin of Replication Complex (ORC) to initiate DNA synthesis
Allows regulated initiation to ensure one round of replication (in about 30 min)
how long would human chromosomes take if they were replicated like bacterial DNA
a month
Eukaryotes with larger genomes have 1 origin of replication
False
How does the cell ensure that the DNA is only copied once?
through Licencing
describe what happens in Licencing
origins of replication can only recruit pre-replicative proteins to origins in G1
Origins can only “fire” DNA replication in S phase, and then are deactivated
what are the components of the DNA replication machine
Helicase
- to separate the DNA double helix
Single-strand binding protein
- to maintain separation of single strands
DNA primase
- to initiate DNA polymerization
Two DNA polymerases
- to synthesise the two new strands of DNA
A sliding clamp (PCNA)
- to keep polymerase on DNA
Topoisomerases
- nick or cut and reseal DNA ahead of the replication fork to remove supercoils and tangles
what is the difference between the leading strand and the lagging strand
DNA strands are anti-parallel
The leading strand can be synthesised continuously
The lagging strand must be synthesised non-continuously as Okazaki fragments
how does proofreading work
The wrong nucleotide can sometimes be added, but “proofreading” activity removes the wrong nucleotide through exonuclease editing
explain the way primers are involved in DNA replication
Proofreading DNA polymerases need a perfectly base-paired nucleotide to add new nucleotides
So, these polymerases cannot initiate new DNA synthesis
They need “primers” from which to extend the new strand
DNA primase creates these primers using RNA
Because the primers are RNA, they can be distinguished from DNA and removed later
DNA ligase then joins the Okazaki fragment
what problem does the need for primers create
how to replicate the end of linear DNA?
The answer is to have a special structure at the chromosome ends: telomeres
In humans, these are repeating units of GGGTTA (approx 1000 repeats)
These repeating units are produced by an enzyme called telomerase
what does telomerase do
Telomerase extends the 3’ end of the chromosome so it can be back filled by lagging strand synthesis without losing genetic information
Telomere binding proteins plus a T-loop structure protect the free end
what pathways repair DNA damage
- Strand-directed mismatch repair
- Base and nucleotide excision repair
- DNA break repair
what does the cell use to repair DNA
Where possible, the cell makes use of the information from the undamaged DNA strand to carry out the repair
In some cases, the cell can even use the information in a sister chromosome to correct damage
describe what happens in Strand-directed mismatch repair
Mis-incorporations during DNA synthesis are not always corrected by DNA polymerase proofreading
Scanning proteins (MutS and MutL) can detect mismatched nucleotides
But, has to be a mechanism to correct only the new strand
In eukaryotes, this happens because only the new nicked strands are repaired
describe what happens in Base and nucleotide excision repair
(A) A number of enzymes (e.g. Uracil DNA glycosylase) recognize different types of damaged base
AP endonuclease and phosphodiesterase remove sugar phosphate
DNA polymerase adds new nucleotide and DNA ligase seals nick
(B) Other enzymes can recognize distortions in the double helix
e.g. excision nuclease recognizes pyrimidine dimers and excises the DNA bit containing it
DNA helicase cuts the excised DNA section
DNA polymerase and DNA ligase make up the new DNA segment
cytosine can deaminate to uracil
True
how does DNA break repair work
Double stranded breaks (DSBs) are a potentially catastrophic form of DNA damage
Can be repaired fairly simply by recognizing and re-ligating the free ends together (NHEJ) but this is error-prone
If cell is in S/G2, then homologous recombination (HR) can use the information in a sister chromosome to accurately repair the damage
how does G2 checkpoint arrest the cell from Mitosis
- Inhibiting M-Cdk through p53 and p21, as described earlier for the G1 checkpoint
- By inhibiting Cdc25, the phosphatase needed to activate M-Cdk
