Lecture 4 - DNA Replication Flashcards
1
Q
Pol A
A
DNA replication
2
Q
Pol B
A
Base excision Repair
3
Q
Pol G
A
Mitochondrial Replication
4
Q
PolDi
A
DNA replication
5
Q
Pol E
A
DNA replication
6
Q
Pol Z
A
Bypass synthesis
7
Q
Pol H
A
Bypass synthesis
8
Q
Pol Q
A
DNA repair
9
Q
Pol I
A
Bypass synthesis
10
Q
Pol K
A
Bypass synthesis
11
Q
Pol L
A
Base excision Repair
12
Q
Pol M
A
Non homologous end joining
13
Q
Pol S
A
Sister chromatid cohesion
14
Q
REVIL
A
Bypass synthesis
15
Q
TDT
A
Antigen receptor diversity
16
Q
In the human genome, how many DNA polymerase enzymes are involved in DNA replication of genome?
A
3
17
Q
What is the size of eukaryotic replicon?
A
40-100kb in length
18
Q
Individual replicons are activated at characteristic times during which phase?
A
S Phase
19
Q
What does regional activation pattern suggest?
A
Replicons near one another are activated at the same time
20
Q
What is key to initiation of replication?
A
Creating the replication fork at the origin oriC
21
Q
What are key elements in initiation?
A
Initiation requires several enzymatic activities:
Helicases
Single-strand binding protein
Synthesis of primers
22
Q
What does initiation at oriC require?
A
Sequential assembly of large protein complex on the membrane
23
Q
Define licensing factor
A
A factor located in the nucleus and necessary for replication that is inactivated or destroyed after one round of replication
New factors must be provided for further rounds of replication to occur
24
Q
Prior to replication
A
Nucleus contains active licensing factor
25
After replication
Licensing factor is in the nucleus (inactive)
| Licensing factor in the cytoplasm cannot enter nucleus
26
What does dissolution of nuclear membrane during mitosis allow?
Licensing factor to associate with nuclear material
27
What does cell division generate?
Daughter nuclei competent go support replication
28
Name the proteins that are licensing factors
MCM proteins
Cdc6
Cdt1
29
What is ORC?
A protein complex that is associated with origins throughout the cell cycle
30
What is Cdc6?
Unstable protein
Synthesised only in G1
Binds to ORC and allows MCM proteins to bind
31
What is Cdt1?
Facilitates MCM loading on origins
32
What happens to Cdt1 and Cdc6 once replication starts?
They leave ORC
33
How does negative regulation of replication occur?
G2 nuclei synthesise Geminin
Geminin sequester Cdt1
MCM proteins are prevented from assembling on freshly synthesised DNA
Geminin is degraded on completion of mitosis
DNA can recommence at the next S Phase
34
Which enzymes are involved in DNA assembly?
DNA polymerases
35
When DNA is synthesised which end are nucleotides added to?
3’-OH end
36
What direction will DNA grow in?
5’ to 3’
37
Nucleophilic attack by the ribose 3’-OH group on alpha phosphate of the incoming nucleotide will have a triphosphate which will release what?
Pyrophosphate (PP) - A diphosphate
38
What is the construction of DNA process?
Primer has 3’-OH end
The incoming nucleotide will have 5’ triphosphate
This will cause the release of diphosphate upon addition
39
What do bacterium or eukaryotic cell have?
Several different DNA polymerase enzymes
40
What are the 2 processes that bacterial DNA polymerase undertakes?
a) semiconservative replication
| b) Repair reactions
41
Which gene codes for enzyme I?
Pol A
42
What is the function of enzyme I?
Major repair enzyme
43
Which gene codes for enzyme II?
Pol B
44
What is the function of enzyme II?
Replication restart
45
Which gene codes for enzyme III?
Pol C
46
What is the function of enzyme III?
Replicase
47
Which gene codes for enzyme IV?
dinB
48
What is the function of enzyme IV?
Translation replication
49
Which gene codes for enzyme V?
umuD’ 2 C
50
What is the function of enzyme V?
Translation replication
51
What is an example of nuclease activity of DNA polymerase?
Bacterial DNA polymerase I had an exonuclease effect
| Combined with DNA synthesis to perform nick translation
52
What is the role of nick translation?
Replaces part of preexisting strand of duplex DNA with newly synthesised material
53
What is the process of Nick translation?
Nick generated 3’-OH
5’p groups DNA synthesis extends 3’end
Old strand is degraded by 5’ to 3’ exonuclease
54
What is high-fidelity DNA polymerases involved with?
Involved in replication
| Have a precisely constrained active site that favours binding of Watson-Crick base pairs
55
Define processivity
The ability of an enzyme to perform multiple catalytic cycles win a single template instead of dissociating after each cycle
DNA polymerases generally require additional factors for processivity
56
What is the common structure of DNA polymerase?
Large cleft composed of 3 domains that resemble a hand
| DNA lies across the palm in a groove created by the fingers and thumb
57
Leading strand
5’ to 3’
58
Lagging strand
3’ to 5’
59
What are the discontinuous short fragments of the lagging strand called?
Okazaki fragments
60
What is semi-discontinuous replication?
The mode of replication in which one new strand is synthesised continuously while the other is synthesised discontinuously
61
What are helicase?
Separate the strands of DNA using energy provided by hydrolysis of ATP
A hexameric helicase moves along one strand of DNA
Encircling it and binding to duplex DNA
Base pairs are separated
The helicase releases duplex
62
What is DnaB?
Helicase that unwinds DNA for replication in E.coli
63
What is MCM?
Helicase that unwinds DNA for replication in prokaryotes
64
What is a single stranded binding protein (SSB) required for?
Maintain separated strands
65
What does all DNA polymerases require?
Free 3’-OH priming end to initiate DNA synthesis
66
What can provide the 3’-OH priming end?
RNA primer
Nick in DNA
Priming protein
67
What is RNAa primer synthesised by?
Primase
68
What is a primase?
A type of RNA polymerase that synthesises short segment of RNA that will be used as primers for DNA replication
69
Where does primase exist?
Complex with DNA polymerase alpha
70
How do leading and lagging catalytic units behave?
Differently
71
In E.coli which enzyme units are required to synthesis the leading and lagging strand?
The same
72
In E.coli which enzyme is required to synthesise the leading and lagging strands?
DnaE - DNA pol III alpha
73
Which three DNA polymerases make up the eukaryotic replication dork?
Alpha
Delta
Epsilon
74
What is DnaB?
Helicase that unwinds DNA for replication in E.coli
75
What is MCM?
Helicase that unwinds DNA for replication in prokaryotes
76
What is a single stranded binding protein (SSB) required for?
Maintain separated strands
77
What does all DNA polymerases require?
Free 3’-OH priming end to initiate DNA synthesis
78
What can provide the 3’-OH priming end?
RNA primer
Nick in DNA
Priming protein
79
What is RNAa primer synthesised by?
Primase
80
What is a primase?
A type of RNA polymerase that synthesises short segment of RNA that will be used as primers for DNA replication
81
Where does primase exist?
Complex with DNA polymerase alpha
82
How do leading and lagging catalytic units behave?
Differently
83
In E.coli which enzyme units are required to synthesis the leading and lagging strand?
The same
84
In E.coli which enzyme is required to synthesise the leading and lagging strands?
DnaE - DNA pol III alpha
85
Which three DNA polymerases make up the eukaryotic replication dork?
Alpha
Delta
Epsilon
86
What is the role of polymerase alpha l/primase complex?
Complex initiates and then primes the synthesis of both DNA strands (approx 10-30 bases)
Synthesises RNA primer and then extends the DNA by 20 bases
Not processive and remains associated with lagging strand
87
What is the role of processive DNA polymerase Epsilon?
Elongates the leading strand
88
What is the role of DNA polymerase delta?
Elongates the lagging strand
89
What is PCNA?
A sliding clamp that helps processivity if polymerase Epsilon and delta
90
What is the clamp loader?
Places the sliding clamp (PCNA) on DNA where they form a circular clamp around the DNA, increases processivity
91
What is an example of clamp loader in eukaryotes?
RFC
92
What is the process of polymerase switching?
DNA synthesis starts with polymerase alpha
Then replaced by the more processive delta and Epsilon
Pol alpha must contact RPA for stable attachment to the primed site
The RFC-PCNA complex competes with pol alpha for RPA
RFC loads PCNA onto DNA but remains associated with PCNA
Pol delta competes with RFC for both RPA and PCNA thereby recruiting PCNA and the 3 primer terminus for chain extension
93
Why is the core on the leading strand processive?
It’s clamp (PCNA) keeps it on the DNA
94
What happens with regards to clamping in Okazaki fragments?
```
The clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragments and reassembles for the next fragment
Core polymerase (pol delta) and the clamp (PCNA eukaryotes, clamp Beta in bacteria) dissociate at completion of Okazaki fragments synthesis and reassociate at the beginning
```
95
What are Okazaki fragment linked by?
Ligase
96
What does synthesis of Okazaki fragment require?
```
Priming
Extension
Removal of RNA primer
Gap filling
Nick ligation
```
97
How does DNA Ligase seals nicks between adjacent nucleotides?
Employing an enzyme-AMP intermediate
