1
Q
central dogma of biology (3)
A
- genetic information moves to proteins
- once information is transferred to protein, it cannot be transferred back to RNA/DNA
- RNA and DNA can pass information back and forth and replicate
2
Q
what are the challenges that need to be resolved to replicate DNA? (5)
A
- DNA copy must be accurate
- must be able to free DNA strands from protein and from each other
- must be repair/proof-reading mechanisms
- must be fast
- eukaryotes only: issue with replicating telomeres
3
Q
telomeres
A
- the ends of linear DNA
4
Q
semi-conservative
A
- describes how DNA molecules contain one strand from original parent DNA molecule and one from newly synthesized strand after replication
5
Q
DNA polymerase (2)
- function
- what does it require
A
- enzyme that uses DNA as a template to make new DNA copies
- requires primer and magnesium 2+
6
Q
DNA polymerase primer
A
- short strand oligonucleotide with free 3’-OH
7
Q
what is magnesium’s function in DNA replication? (2)
A
- acts as a co-factor for DNA polymerase by coordinating dNTP in the active site
- aspartate forms ionic bond with magnesium in the active site
8
Q
what direction does DNA polymerase catalyze the synthesis of new DNA?
A
- ONLY in the 5’ -> 3’ direction
9
Q
fidelity
A
- the degree of exactness with which something is copied or reproduced
10
Q
how does DNA polymerase achieve high fidelity (3)
A
- Mg2+ ensures proper orientation of dNTP
- geometry of active site ensures only correct base pairing can occur
- alpha-helix “lid” closes on active site when correct base pairing forms to trap dNTP so catalyses can occur
11
Q
how much does magnesium, active site geometry and alpha-helix allow mistakes in DNA?
A
- allows a mistake every 10^4 - 10^5 nucleotides
12
Q
how does DNA polymerase correct mistakes that do occur?
A
- 3’ -> 5’ exonuclease activity removes nucleotides one by one from DNA strand
13
Q
what are the steps involved in DNA polymerase’s exonuclease activity? (3)
A
- if mismatch base is incorporated, DNA polymerase will shift to 3’ -> 5’ exonuclease site
- mismatch is removed
- DNA polymerase will switch back to 5’ -> 3’ active site and continue polymerase activity
14
Q
what is a downside of DNA polymerase exonuclease activity and why is it still used anyway? (2)
A
- it is wasteful as it can remove pairings that are not mismatched
- still used because it does ensure high fidelity
15
Q
how much does DNA polymerase exonuclease activity allow mistakes in DNA?
A
- allows a mistake every 10^6 - 10^8 nucleotides
16
Q
what happens if the exonuclease activity fails to detect a mismatch?
A
- DNA repair enzymes can correct error after replication
17
Q
what is the final error rate considering all of the mechanism used to ensure high fidelity in DNA replication?
A
- a mistake every 10^10 nucleotides
18
Q
describe the E.coli genome
A
- circular genome with one oriC
19
Q
oriC definiton
A
- origin of replication
20
Q
oriC structure (2)
A
- 4-5 repeats of a sequence recognized by DnaA protein
- DNA unwinding elements (DUE)
21
Q
DNA unwinding elements (DUE) (2)
A
- 3 tandem repeats (~13 nucleotides each) rich in A and T
- where first strand separation occurs due to weaker H-bonding (only 2 bonds) between A-T
22
Q
replication fork
A
- point where DNA is being synthesized
- replication occurs bidirectionally: 2 replication forks per circular genome
23
Q
does DnaA require ATP (2)
A
- yes, it required ATP
- ATP is a co-factor for DnaA because it is not hydrolyzed
24
Q
DnaB (2)
A
- helicase: separates the DNA helix
- one DnaB per replication fork, so there are 2 DnaB proteins total during DNA replication
25
DnaC
- ATPase: it hydrolyzes 1 ATP to load DnaB
26
how does DnaB bind to the replication fork?
- with the help of DnaC, which hydrolyzes an ATP
27
what Dna proteins stay/leave the DNA helix? (2)
- DnaB stays in front of DNA polymerase in each replication fork to separate strands
- DnaC and DnaA dissociate from DNA after initiation
28
what enzyme is located in front of DnaB during DNA replication? (2)
- function
- topoisomerase, specially DNA gyrase
| - remove supercoils formed by DNA unwinding the helicase/DnaB
29
ss binding proteins
- bind to ssDNA during DNA replication to prevent DNA renaturation
30
leading strand (2)
- synthesized continuously
| - synthesized in direction that DNA unwinds
31
lagging strand (2)
- synthesized discontinuously in short stretches
| - synthesized in the direction opposite to DNA unwinding
32
okazaki fragments
- short stretches of DNA synthesized on lagging strand
33
DNA polymerase I (3)
- where is it found
- different activities
- main function
- DNA polymerase found in E. coli
- has 3 activities: polymerase, proof-reading (3' -> 5' exonuclease), and 5' -> 3' exonuclease
- nick translation: using 5' -> 3' exonuclease it removes RNA primers and substitutes them with DNA
34
what does each synthesized DNA segment start with?
- RNA primer
35
what must be done before Okazaki fragments can be stitched together
- RNA primers must be removed
36
how are RNA primers removed form Okazaki fragments (2)
- DNA polymerase I 5' -> 3' exonuclease activity removes nucleotides in front of moving enzyme
- results in nick translation
37
nick translation
- due to removal of RNA primer by DNA polymerase I, ss break in DNA will be moved further down the strand
38
function of DNA polymerase I 5' -> 3' exonuclease activity (2)
- removal of RNA primers from Okazaki fragments
| - DNA repair
39
DNA polymerase III (4)
- where it is found
- physical characteristics
- function
- physical features and characteristics
- DNA polymerase found in E. coli
- large protein with many polypeptide chains
- one part of enzyme synthesizes leading strand while another part synthesizes lagging strand at the same time (2 active subunits at the same time) using RNA primers as a starting point
- has high fidelity and processivity due to beta-clamp and clamp-loading complex
40
how does DNA polymerase III have high fidelity and processivity
- it can catalyze many reactions without releasing the substrate (DNA)
41
beta-clamp (2)
- made of 2 beta-subunits that can close on DNA to allow DNA polymerase III to have high processivity
- 1000 nt/s vs 10 nt/s in DNA polymerase I
42
clamp-loading complex
- δ-loader that can open beta-clamp and thread DNA through
43
what DNA polymerases are used in E. coli
- DNA polymerase I and III
44
what primase is used in E. coli for DNA replication
- DnaG
45
DnaG (2)
- primase used for DNA replication in E. coli
| - synthesizes RNA primers (4-5 nt long) on both leading and lagging strand
46
describe discontinuous synthesis in E. coli DNA replication
- every 1000-2000 bp, DNA polymerase III releases lagging strand of DNA and binds it further down the replication fork where primase has synthesized a new primer
47
does clamp loading require ATP?
- yes, it requires 3 ATP molecules
48
DNA ligase (2)
- connects ss breaks and seals nicks between Okazaki fragments
- requires 1 ATP (2 ATP equiv) molecule per nick
49
ter sites
- special sequences in E. coli that are located opposite to the oriC
50
Tus protein
- bind to ter site sequences and "slow down" the replication fork
- 2 sets of Tus-ter complexes
51
role of topoisomerase in E. coli DNA replication
- topoisomerase IV resolves catenated DNAs after termination
52
what is happening during the same time as DNA replication in prokaryotes
- cell division
53
how does replication in eukaryotes differ from replication in prokaryotes (9)
1. more complex and not fully understood
2. larger genomes
3. linear chromosomes
4. movement of replication fork is slower
5. presence of histones
6. multiple origins of replication per chromosome
7. okazaki fragments are shorter
8. different main polymerase enzyme used
9. primers made and removed by different enzymes
54
how much slower is the movement of the replication fork between eukaryotes and prokaryotes (2)
- why is it slower in eukaryotes
- 50 nt/s vs 1000 nt/s
| - mistakes are more detrimental in eukaryotes
55
how much shorter are Okazaki fragments between eukaryotes and prokaryotes
- 100-200 nt vs 1000-2000 nt
56
what is the main enzyme of replication in eukaryotes?
- polymerase δ
57
what makes RNA primers in eukaryotes
- primase: polymerase alpha
58
what removes RNA primers from Okazaki fragments in eukaryotes?
- combination of RNase H and MF1
59
RNase H
- degrades RNA-DNA duplexes
60
MF1
- has 5' -> 3' exonuclease activity
61
what problem does DNA polymerase δ have with replicating telomeres
- DNA polymerase δ can only synthesize in 5' -> 3' direction, so DNA telomeres are left unreplicated after primer is removed
- ss overhang is degraded so DNA gets shortened
62
telomere
- linear chromosome ends
63
what is the solution to replicating telomeres?
- telomerase enzyme uses reverse transcription
64
telomerase (2)
- special enzyme in eukaryotes capable of reverse transcription
- can extend DNA ends by using RNA as a template
BIOC 302 Part 2
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