Apricio - Lecture 1-5 Flashcards
(38 cards)
1
Q
Arthur Kornberg
A
- isolated a protein enzyme; DNA polymerase I (Pol I)
- dNTP are required precursors (building blocks) of DNA
- template DNA required for DNA synthesis
- DNA Pol I and dNTP and template required
- He showed that newly synthesized DNA molecules had a similar base composition = using templates with different base composition
2
Q
Three models for the replication of DNA
A
- distributive
- semiconservative
- conservative
3
Q
Kornberg; mechanism by which daughter DNA molecules were assembled
A
Kornberg’s experiment established that strand of DNA served as a template for DNA synthesis; but not yet revealed the mechanism how daughter DNA molecules were assembled
4
Q
DNA Polymerase I
A
Enzymatic Synthesis of DNA
- DNA polymerase removes pyrophosphate
- pyrophosphatase breaks the bond between phosphate groups
5
Q
Requirements for DNA synthesis
A
- single stranded DNA template
- annealed (complementary) primer with a 3’-OH
- dNTPs
- DNA Polymerase
6
Q
Mechanisms of DNA synthesis
A
- Correct base pairing allows the reaction to take place (by properly positioning reactive groups in the polymerase active site)
- Hydrolysis of pyrophospate drives the reaction
* 3’OH on primer attacks (catalysis) the link between phosphate alpha and beta
* Structure dictates function
7
Q
DNA Polymerase resembles a RIGHT HAND
A
- only close on correct base-pair (two purine is too big, two pyrimidine is too small)
- catalyze the reaction when correct base-pair (closely adjust)
- structure allows enzyme to repeat the catalytic cycle (hold on to the DNA)
- Metal ions used to shield negative charges of dNTPs
- Metal ions used to activate the reactive 3’ OH
8
Q
DNA Polymerase = processive enzymes
other enzymes work distributed
A
PROCESSIVITY: ability of DNA polymerases to caarry out continuous DNA synthesis without dissociating from the template
== greatly increases the rate of DNA synthesis
* DNA polymerase binding is slow
* DNA synthesis is fast
9
Q
DNA Polymerase & proofreading exonuclease (“delete” key)
A
DNA Polymerases contain proofreading exonulease
- incorrect nucleotide polymerased into DNA (incorrect structure) will inhibit further synthesis - very rare (1/100000)
- misshapen DNA moves from (polymerase active site) to (second enzymatic site) on protein that contains “3’ to 5’ exonuclease activity”
- proofreading exonuclease removes the terminal 3’ base = restore correct geometry of primer-template junction
- DNA moves back to polymerase site
10
Q
Replication Fork :: Opposite Directions of DNA Synthesis
A
DNA unwinding creates a fork structure
- Fork structure moves as more DNA is unwound
- Both (two single strands) DNA strands act as templates for DNA synthesis
- DNA strands are antiparallel & DNA synthesis ALWAYS occurs 5’ to 3’ ==> two strands replicated in opposite directions relative to the movement of the replication fork
* leading strand and overall direction of DNA matches
* lagging strand polymerase move opposite direction (form Okazaki fragments)
11
Q
Leading and Lagging strand DNA synthesis
A
- Leading strand: 1. replicated in same direction as the fork 2. one long continuous strand of nascent DNA
- Lagging strand: 1. replicated in opposite direction 2. synthesized discontinuously as Okazaki fragment (100s ~1000s)
12
Q
Primase: primer is synthesized by primase
A
Primase:
- RNA polymerase; can begin synthesis without a primer DNA polymerase requires a primer
- begins DNA replication by synthesizing 5-10 base RNA molecule complementary to the template
- DNA polymerase use 3’OH of RNA primer to continue DNA synthesis
13
Q
dNTPs
A
deoxyribonucleocide triphosphates
doxyriboNocleocide TriPhosphates
14
Q
template primer junction
A
template DNA and annealed primer
15
Q
gap in DNA strand
A
missing a base
16
Q
nick in DNA strand
A
break of phosphate bond between bases
17
Q
For Konberg to verify his findings, he was lucky to have DNA that are not pure DNA single strands
A
DNA he threw in had “nicks” in it
18
Q
E. coli has 5 polymerases (10 in human)
A
Its chromosone is duplicated in 30 minutes
19
Q
DNA synthesis direction and DNA reading direction
A
DNA synthesis: 5’ to 3’
DNA reading: 3’ to 5’ (direction of polymerase movement)
20
Q
Removal of RNA primer
A
- RNAseH: (endonuclease) remove RNA backbone; recognizes DNA-RNA hybrids
- 5’ exonuclease: removes last ribonucleotide of RNA primer
- DNA polymerase: extend 3’OH (at new primer-template junction)
- DNA ligase: joins the DNA using 5’PO_4 and 3’OH at the nick
21
Q
DNA ligase
A
- works similar to topoisomerase 1
2. use ATP since no E is stored in DNA
22
Q
5’ exonuclease is not same as proofreading exonuclease
A
- 5’ exonuclease removes 5’ end
2. proofreading exonuclease (or 3’ to 5’ exonuclease) removes 3’ end
23
Q
DNA Helicase
A
- multi subunit protein - hexamers form donut shape; encircle DNA(single strand); keep bound (processive) and move in one direction (polarity; 5’ to 3’)
- unwind DNA for replication (and/or transcription) - use energy of ATP (a lot; 1 ATP per 2 bases)
24
Q
DNA unwinding causes DNA supercoiling
A
- helicase–> positive supercoil ahead, negative supercoil behind
- Toposiomerase type 1 and 2 remove positive supercoils; type 2 is necessary
25
Single-Stranded Binding Protein (SSB)
1. keep DNA unwound for DNA synthesis; required for lagging (slow) strand; to prevent intramolecular base-pairing);
2. binding is sequence independent;
3. binding happens immediately
4. cooperative manner (binding of one protein facilitates the binding of the next)
5. SSB binding prevents intramolecular base-pairing
6. SSB bidning keeps the DNA in extended conformation with bases exposed to facilitate synthesis
26
Different polymerases removing RNA primer
Pol III: cannot act at a nick; dissociates so that Pol I can come in
Pol I: perform either 1. strand displacement 2. Nick translation
* Nick in the DNA remains
27
Strand displacement
performed by Pol I
| "remove 5' nicked end intact" as Pol I simultaneously synthesizes DNA on exposed template
28
Nick translation
involves "degradation of nicked strand from its 5' end - 5' to 3' exonuclease" with simultaneous synthesis of DNA on exposed template
* Eukaryotic polymerases do not carry out nick translation
29
Processing lagging strand in Eukaryotes (dealing with RNA primers in Eukaryotes)
Pol-delta displaces RNA primer & some DNA
===> produce flap ==> flap cleaved by Fen1 (flap endonuclease 1)
*Fen1 only works on DNA; so some of DNA will also be cut off
** Nick in DNA is still not removed
30
Prokaryotes: Multiple DNA Polymerases function in DNA replication
Pol III: carries out most synthesis(leading & lagging)
| Pol I : completes lagging strand synthesis
31
Eukaryotes: Multiple DNA Polymerases function in DNA replication
1. Pol alpha: takes over from primase; has limited processivity; less accurate than Pol delta, Pol epsilon
2. Pol delta & Pol epsilon: replicate most of the DNA; has sliding clamp
a. Pol delta : lagging strand
b. Pol epsilon: leading strand
32
sliding clamps
1. increase processivity of DNA polymerases
a. beta: prokaryotic clamp; 2 subunit
b. PCNA: eukaryotic clamp; 3 subunit
2. opened up by "Clamp-loader" protein complex between subunits; requires ATP binding and hydrolysis (coupled to the last step; no energy wastes) to load the clamp
3. DNA polymerase released from sliding clamp in the absence of a primer.. (after completion of an Okazaki fragment)
33
DNA sequencing and Dideoxynucleotides
- Dideoxynucleotide lack 2'OH and 3'OH
| - dideoxynucleotide in DNA blocks further polymerization because it lacks 3'OH
34
DNA sequencing by Chain Termination
- use Dideoxynucleotides
1. (radioactive) primer is annealed to DNA molecule of interest (preceding sequence must be known)
2. presence of all four dNTPs & DNA polymerase, primer will be extended to end of the template
3. additional presence of tiny bit of didexoynucleotide, chains will stop growing when dideoxynucleotide is incorporated (rarely happens)
4. then position of complementary base can be predicted
35
DNA sequencing gel
1. Four different reactions are performed (each with different dideoxynucleotide: ddA, ddC, ddT, ddG)
2. reaction run on a denaturing polyacrylamide gel; allows analysis of the single synthesized strand with single base resolution
3. primer is radioactive; visualized on the gel
4. position of band indicates where each base was incorporated
36
DNA sequencing by High-Throughput DNA Sequencing
* sequencing reactions are carried out in large batches
- sequencing carried out all together in a single reaction with all four normal dNTPs and low concentration of each dideoxy-NTPs (each labeled with different fluorescent-colored dye)
- colored DNA molecules detected as passing the end of column, can read out the sequence
37
Molecular requirements for PCR
PCR: polymerase chain reaction
1. template DNA
2. primer pair complementary to before and after sequences of the target
3. DNA polymerase (thermostable)
4. dNTPs
38
Use of PCR
- amplification of large amounts of specific or random DNA from a small sample
1. cloning (mole. level)
2. making probes (for southern/northern blots)
3. gene mapping
4. mutagenesis
5. diagnostics/forensics
* primers become part of the product
