1
Q
list Phases of the cell cycle in order
A
G1 —- if cell cycle check points not met then G0 phase
S
G2
M
2
Q
G1 Phase
A
- cell grows and preps for cell division
- check if previous cell cycle was completed properly
- checks that growth and maturation have been completed to the appropraite levels
—- If cell cycle checkpoints not approved - cell goes into non-dividing phase - G0 phase (stops growing here)
—– remain in G0 for extended periods of time
3
Q
S phase
A
DNA replication phase
4
Q
G2 phase
A
- cell preps for mitosis by:
– duplicating organelles
– growing
– producing relevant proteins - after specific maturation point reached – cell progresses into M phase
5
Q
M phase
A
- Mitosis and cell division phase
- after DNA replication – condensed into chromosomes
- chromosomes move to opp poles of cell and cell divides
6
Q
Three stages of DNA rep
A
Initiation, Replication/DNA synthesis and termination
7
Q
where does decision to replicate occur and why?
A
at the initiation point – once DNA replication begins it goes till the end
8
Q
points on DNA where replication begins called?
A
ori - origins of replication
9
Q
ori in prokarys and eukarys
A
prokary - 1 ori (since circular DNA)
eukary - multiple oris
10
Q
how many times can initiation of replication happen per cell cylce and why
A
ONCE - multiple times would lead to cell death as wrong number of genomes would occur in a cell
11
Q
Precursors of DNA
A
12
Q
how many high energy phosphate bonds in dNTP
A
13
Q
DNA synthesis enzyme
A
14
Q
primer
A
15
Q
direction of dna rep/synthesis
A
5’-3’
DNA polymerase can only attach an incoming dNTP onto the 3’ OH group of a deoxyribose
16
Q
what does DNA polymerase catalyses the formation of
what is consumed and released in this reaction
A
a phosphodiester bond btwn the phosphate on the 5’ carbon of an incoming dNTP and the 3’ OH on the primer
generating a 5’-3’ phosphodiester bond.
consumed - high energy dNTP
released - pyrophosphate (PPi)
17
Q
-What is the basic DNA polymerase error rate?
A
In vitro is 1 mistake per 100,000 new bases incorporated
Error rate is 1 x 10^-5
However actual number after 1 round is 1 x 10^-10
18
Q
ways errors are reduced in DNA synthesis
A
Proofreading by DNA poly - during DNA synthesis - reduces error by 100 fold
MMR (mismatch repair) - after DNA synthesis - further reduces error by 100 fold
19
Q
Proofreading basic concepts
A
- Occurs during DNA synthesis
- Done by a 3’-5’ exonuclease activity in DNA poly.
- 3’-5’ exonuclease activity – digests DNA starting at 3’ end and moves towards the 5’ end
- If a nucleotide is misincorporated then:
— Polymerisation by DNA pol. slowed down by 10,000 fold
— Activity of 3’-5’ exonuclease increases - Exonuclease excises wrong nucleotide and a few additional nucleotides
- DNA pol. then resynthesises this excised section and continues synthesis
20
Q
Exonuclease activity direction
A
3’-5’ exonuclease activity – digests DNA starting at 3’ end and moves towards the 5’ end
21
Q
mismatch repair (MMR) process
A
- About 1 time per 100 – misincorporated nucleotide not recognised by DNA pol. proofreading system
- thus remains in DNA after replication - creating mismatched base pair
- Mismatched base pair recognised by MMR system repair and is corrected
- The mismatch repair system needs to be able to distinguish the old strand of the DNA (templates strand) from the newly synthesised strand to be able to replace the mismatched DNA base from the new DNA strand.
22
Q
the number of new mutations per daughter
cell is?
A
1
Approximately 1 new mutation is introduced in
each cell division in humans.
23
Q
Why is every cell different
A
Human has 10^14 cells
about 10^17 cell divisions occur in humans in their life
everytime cell divides - gets one new mutation
- thus each cell is a little different from the other
24
Q
what can defects in MMR cause
A
HNPCC - Hereditary NonPolyposis Colorectal Cancer
previously called lynch syndrome
25
Amsterdam criteria for HNPCC
- 3 relatives over 2 gens
- 2 must be first degree relatives
- 1 must be under 50 at the time of diagnosis
- FAP ( family adenomatous polyposis) must be excluded
26
incidence of HNPCC
2-5% of the population
15% of men under 50 w/ colorectal cancer
27
what can cause HNPCC
- mutations in any of the mismatch repair genes for the proteins that recognise different forms of mismatched base pairs in DNA can cause HNPCC
28
Key initiator proteins in E.coli and eukaryotes
E.coli - dnaA
Eukaryotes - ORC
29
function of helicase
unwinds the DNA
forms a replication bubble at ori
30
SSB protein function
Single Stranded Binding protein -- Binds to single stranded DNA and keeps it single stranded
31
what does DNA pol need to bind to start synthesis
An RNA primer with a free 3' -OH group
32
what synthesises the RNA primers used by DNA pol in dna synth.
DNA primase
A RNA primer is synthesised at the ori by a protein called DNA primase
33
how is supercoiling relieved in humans and prokaryotes
humans -- Type I and II Topoisomerases
Prokaryotes -- Type II Topoisomerase called DNA gyrase
34
steps of intitiation of dna synthesis in order
1. Recognition ---- by dnaA and ORC in E. coli and eukaryotes respectively
2. Melting ---- of DNA at ori so proteins can bund
3. Unwinding ---- of double strand by helicase + replication bubble -- SSB proteins to keep DNA sungle stranded
4. Recruitment ---- by the proteins at ori of more proteins that will actually start dna rep.
5. Primer --- attache at ori synthesised by DNA primase -- supercoiling in DNA relived by topoisomerase I and II in humans and a type of topoisomerase II in prokaryotes called DNA gyrase.
6. DNA synthesis - in 5' to 3' direction begins
35
proteins involved in initiation of DNA replication and the function of each protein.
DnaA -- binds to ori - key initiator protein in E.coli
ORC -- binds to ori - key initiator protein in eukarys
Helicase -- unwinds DNA - replication fork
SSB proteins -- keeps single stranded DNA single stranded
Melting proteins : Melt DNA at ori
DNA primase -- synthesises RNA primers
Topoisomerase I and II -- uncoils DNA -- opens up supercoiling of DNA -- in humans
DNA gyrase (type of topoisomerase II) -- -- uncoils DNA -- opens up supercoiling of DNA -- in prokaryotes
DNA polymerase: Synthesis DNA stranded
36
---Explain leading and lagging strand DNA synthesis.
Leading:
DNA polymerase starts synthesis from the (RNA primer which is synthesised from the ori by DNA primase)
Goes from primer and goes all the way to the end of the DNA molecule, and this newly made DNA strand is called the leading strand
Made by DNA polymerase III in E. coli
Made by DNA polymerase epsilon in humans
Lagging:
Synthesised by Okazaki fragments -- DNA synthesis occurs in both directions -- one going 5' to 3' direction and other going 3' to 5'
3' to 5' not possible , DNA synthesis can only processed in 5' to 3'
Lagging strand overall goes in 3' to 5' but synthesis is still in 5'-3' -- made up of small Okazaki fragments ,starting from RNA primers
RNA primers -- Made by DNA primase and DNA polymerase I in E. coli or DNA polymerase delta in humans
RNA primer removed by:
in E. coli -- 5' - 3' exonuclease activity of DNA polymerase I in humans-- by FEN-1 and RNAseH
Fragments ligated by:
DNA ligase - in prokaryotes
DNA polymerase - in humans
37
---List the proteins involved in leading and lagging strand DNA synthesis and the function of each protein.
38
List the function of the following DNA replication enzymes:
a)DNA polymerase I,
b)DNA polymerase III,
c)DNA helicase,
d) DNA ligase,
e)DNA primase,
f)DNA topoisomerase
39
---What is the difference between leading and lagging strand DNA synthesis?
40
List the names and function of the enzymes involved in E. coli DNA replication (Enzyme summary E. coli DNA replication)
unwinding of DNA helix: DNA helicase
primer synthesis: DNA primase
leading strand synthesis: DNA polymerase III
lagging strand synthesis: DNA polymerase III
relieving supercoiling in front of replication fork: DNA gyrase
proofreading: DNA polymerase I and III (3’-5’ exonuclease)
primer removal: DNA polymerase I (5’-3’ exonuclease)
proofreading is carried out by DNA polymerase I (3’-5’ exonuclease) for any section of DNA that it synthesises
sealing the nicks: DNA ligase.
41
What is the difference between DNA polymerase I and DNA polymerase II?
DNA polymerase III:
Leading strands are synthesised by DNA polymerase III in E. coli
Start from initiation point , in a 5' to 3' direction
Synthesis is in the 3' to 5' direction however overall
Small DNA fragments called okasaki fragments in 5' to 3' direction are extended by DNA polymerase III in E. coli
DNA polymerase III also removed the RNA primers of Okazaki fragments
DNA polymerase I:
In E. coli , DNA polymerase I binds to 3' end of Okazaki fragment
Extends fragment by synthesising new DNA from 3' end, till it meets an RNA primer
It has 5'-3' exonuclease activity ,which it uses to degrees the RNA primer in front whilst continuing to synthesise DNA behind it
DNA poly I dissociates when RNA primer fully degraded and DNA synthesis completed as far as possible from previous primer
42
-What is the difference between DNA primase and DNA ligase?
DNA primase:
Makes the DNA primer of 20-30 RNA bases
DNA ligase:
Binds the ends of DNA from okasaki fragments together
43
What is the difference between DNA helicase and DNA topoisomerase!
DNA helicase:
Unwinds the DNA
DNA topoisomerase:
Relieves coiling that builds up in front of replication fork , due to unwinding of DNA in eukaryotes
44
List the names and function of the enzymes involved in human DNA replication (Enzyme summary humani DNA replication)
unwinding of DNA helix: ORC / DNA helicase
primer synthesis: DNA polymerase alpha / DNA primase
leading strand synthesis: DNA polymerase epsilon (5’-3’ pol)
lagging strand synthesis: DNA polymerase delta (5’-3’ pol)
relieving supercoiling in front of replication fork: DNA topoisomerase I and II
proofreading: DNA polymerase delta / epsilon (3’-5’ exonuclease)
primer removal: Fen-1, RNAseH
primer extension after primer removal: DNA polymerase delta (5’-3’ pol)
sealing the nicks: DNA ligase I
Inhibition of topoisomerase II kills the cell and inhibitors of
topoisomerase II are widely used as anti-cancer agents.
Inhibition of DNA gyrase kills the cell and some antibiotics work by inhibiting DNA gyrase.
45
what are widely used as anti cancer agents
inhibitors of topoisomerase II
Inhibition of topoisomerase II kills the cell and inhibitors of
topoisomerase II are widely used as anti-cancer agents.
Inhibition of DNA gyrase kills the cell and some antibiotics work by inhibiting DNA gyrase.
46
Termination of DNA replication - E. coli
- genome of E.coli - circular
- replication fork proceeds around the circle to the termination site
- Termination site opposite the ori -- contains 10 ter sequences
- Tus protein binds to 10 ter sequences
- Protein action of Tus along with other proteins completes replication of the circular genome
- The replicated nomes are interwined (catanated)
- Catanated genomes resolved/separated by the Topoisomerase IV protein
47
how many tus sequences at the termination site
what binds to these
what separates the catanated genomes after replication
10 ter sequences
the tus protein
Topoisomerase IV
48
Termination of replication of DNA in eukaryotes and problems associated with it
- Very diff from prokaryotes - as genome is linear and not circular
- DNA on leading strand -- can be synthesised till the end of the chromosome
- Lagging strand synthesis - is problematic at the end of the chromosome -- DNA pol. needs a primer w/ free 3' -OH grp to synthesise DNA -- RNA primers cant remain in DNA -- If removed -- chromosomes would have a gap at the end and thus shorten each time replication occurred
49
The key elements of a chromosome
- one or more oris
- two Telomeres
- one centromere
50
what is located at the ends of chromosomes
telomeres
The ends of chromosomes have specialised structures called telomeres
51
What are telomeres? What is the structure of a telomere?
- Ends of chromosome have telomeres
- They are a large nucleoprotein complex
- Very stable -- protect the ends of chromosomes from damage/degredation
- "caps" ends of chromosomes and prevents it from fusing with other chromosomes (chromosome fusion -- major abnormalities)
- DNA in telomeres consists of multiple short repeating units -- TTAGGG repeated 1000s of times
- Telomerase maintains the length of the telomeres
52
What maintains length of telomeres
telomerase
53
repeating sequence in telomeres
TTAGGG -- 1000s of times
54
function of telomerase
Two main components of human telomerase
Maintains length of telomere
TERT protein -- catalytic protein component (Telomerase Reverse Transcriptase)
RNA component TERC (Telomerase rna componenet) -- has a template (5’-CUAACCCUAAC -3') -- to add
TTAGGG to the 3’ end of telomeres and several accessory
proteins.
55
Explain how telomerase replicates the ends of chromosomes
IN ESSENCE - telomerase adds 6 bases to the 3' end of the chromosome end n a repeated fashion
- Binding: telomerase binds to the 3' end of the chromosome
- Elongation -- Telomerase uses a sequence on its own RNA to extend as a template for extending the 3' end of the chromosome -- it extends it by 6 bases -- GGTTAG
-Translocation -- telomerase then translocates the 6 bases and repeats the process and so on
**number of TTAGGG repeats at end of chromosomes increases with telomerase action
**REVERSE TRANSCRIPTASES -- enzymes which synthesise DNA on an RNA template are called this -- telomerase does this
56
Reverse transcriptases
Enzymes which synthesise DNA on an RNA template
example -- Telomerase
57
where is telomerase expressed and where is it repressed
repressed - somatic cells
expressed - germ cells, stem cells and cancer cells
58
Describe the relationship between telomerase and ageing and cancer cells
- Telomere length and cellular senescence have a strong correlation
-- Telomerase repressed in somatic cells
-- thus telomere repeats lost every mitosis
-- eventually telomere shortening reaches a threshold - cell death
-- reintroduction of telomerase into fibroblasts lengthens their telomeres - extends mitosis cycles they can go thru before cell death
-- telomere shortening -- mitotic clock in normal cells
Cancer cells:
- telomerase is expressed in germ cells, stem cells and cancer cells thus have a greater replicative capacity than somatic cells
- Telomerase is activated in >85% of cancer cells and and telomere lengthening is strongly correlated with cancer
- Telomerase is a good target for an anticancer agent
59
main types of RNA viruses
Two main types:
a. viruses that copy their RNA genome into new RNA
(RNA genome --> new RNA)
b. viruses that copy their RNA into a DNA intermediate which is then used to produce new RNA.
(RNA genome --> DNA intermediate --> new RNA)
60
viruses that copy their RNA genome into new RNA
classified into:
- double stranded RNA viruses
- +ve strand RNA viruses -- RNA can be directly translated into protein
- -ve straand RNA viruses -- RNA that must be
converted into +RNA for translation into proteins
- These viruses encode their own polymerase for replication called RNA dependent RNA polymerase.
61
Viruses that copy their RNA into DNA
- They are retroviruses
- have +RNA.
- copied into DNA by viral encoded reverse transcriptase. - - The DNA is then inserted into the genome of the
host and the inserted genome is transcribed into +RNA by the host RNA polymerase (eg. HIV is a retrovirus).
62
why do new viral strains arise so rapidly
-RNA pol. does not have proofreading capacity
- thus MMR canot be carried out on RNA genome
- Thus mutation rate in RNA viruses is very high
- Thus high mutations - new viral strains produced rapidly
63
what type of virus is Hep C
+RNA virus
64
What is a silent mutation, a missense mutation and a none sense mutation
Silent mutation: mutation still codes for the AA, usually no effect
Missense: have major to minor effect to no effect , changes AA codon
Nonsense : major effect as it changes the codon to a STOP codon, cause proteins syn to stop early, leading to truncated protein, usually kills function of the protein
65
nhibitors of DNA replication
Nucleotide triphosphate inhibitors
Antimetabolites
Inhibitors of chain elongation
DNA polymerase inhibitors
DNA damaging drugs
DNA topoisomerase inhibitors
66
Why is AZT an effective anti-retroviral agent? Why does it not affect retroviral replication but not human DNA replication ?
- used to treat HIV infections
- reverse transcriptase of HIV -- higher affinity
for AZT than dTTP
- human DNA polymerases -- higher affinity
for dTTP than AZT.
- Thus AZT can be used selectively against HIV reverse
transcription of RNA into cDNA without affecting the
DNA polymerases of the host cell.
67
What is the effect of the following on DNA replication : methotrexate , 5-flurouracil , AZT , cisplatin , etoposide?
Methotrexate - antifolate , prevent formation of pyrimidnes/purines
5-flurouracil - pyramidine antimetabolite
AZT- inhibitor of chain elongation
Cisplatin- DNA damaging drug
Etoposide - DNA topoisomerase inhibitor
