1
Q
Who articulated the central dogma of molecular biology first?
A
Francis Crick in 1958
Nobel prize in 1962 to Watson, Crick and Wilkins for discovery of DNA structure
(not to R Franklind b/c she died in 1958)
2
Q
What does the central dogma outline?
A
Process of
1) DNA replication
2) DNA transcription
3) mRNA translation
3
Q
What are the two main parts of the central dogma?
A
1) the replication of DNA that is passed on to two identical daughter cells
2) transcription and translation of information stored in DNA to make RNA and/or proteins = expression of genes/control of phenotype
4
Q
Are all RNAs translated into proteins?
A
No, some RNAs remain as RNA and can function that way
5
Q
What is reverse transcription?
A
A specialized process that utilizes an enzyme (reverse transcriptase) to copy RNA information back into DNA
6
Q
Where is a lot of reverse transcriptase found?
A
A lot of reverse transcriptase enzyme is found in retroviruses
7
Q
Why is DNA replication important?
A
- to ensure exact copy of species’ genetic information is passed from cell to cell during growth & from generation to generation
- if DNA didn’t replicate, life could not continue
8
Q
What are the complementary base pairs of DNA?
A
A-T and G-C
9
Q
Describe the hydrogen bonding in the base pairs of DNA
A
3 hydrogen bonds connect G with C
2 hydrogen bonds connect A with T
10
Q
How many base pairs are there in each turn DNA?
A
10 base pairs per turn
11
Q
How many nm are there between stacked bases and per helical turn of DNA?
A
- 0.34 nm between stacked bases
- 3.4 nm per helical turn
12
Q
Describe the structure of DNA in detail
A
- anti parallel 5’ to 3’ DNA strands
- right-handed double helix (B-DNA: most common form in living cells)
13
Q
If a linear double-stranded DNA is 10,000 bp long (10kb), how many complete turns of the double helix are there? And what is the length of the molecule in micrometer?
A
Complete turns:
DNA has 10 bp per turn, therefore 10000/10 = 1000 turns
Length in micrometer
There are 3.4 nm in each of the 1000 turns = 3400 nm or 3.4 micrometer
14
Q
How many phosphorus atoms are there if there is one phosphorus atom per nucleotide in a DNA strand with 10,000 bp?
A
Each nucleotide has one phosphorus atom, there are 10000 nucleotides on each strand, so 20,000 in total and each has 1 phosphorus atom
Therefore, 20,000 phosphorus atoms
15
Q
If there is 30% A in a double-stranded helix, then how much G is there? How much if DNA were single-stranded?
A
- if 30% A, then 30% T, leaves 40% to be split between G and C
Therefore, 20% G
16
Q
Which double-stranded DNA would be more stable: GC rich or AT rich?
A
GC rich DNA is more stable because it has a greater number of H bonds holding bp together
17
Q
How many hydrogen bonds are there in 5’ GATC 3’, 3” CTAG 5’?
A
GC have 3 AT have 2 TA have 2 CG have 3 Therefore: 10 H bonds
18
Q
What is meant when DNA replication is said to be semi-conservative? (First replication)
A
- during replication each of the original parental DNA strands is a template for the production of a new, complementary daughter DNA strand
- this means that at the end of replication each strand consists of ONE of the original parental strands and ONE of the new strands, i.e. half is conserved in each daughter cell (semi-conservative) after ONE replication
19
Q
What is dispersive replication?
A
- a model considered in the late 50ies
- in both the first and second round of DNA replication, none of he original parental double helix is preserved, rather old and new strands are chopped up and combined so each strand has segments of OG parental AND new strand (hybrid)
20
Q
What happens during the second replication in the semi-conservative DNA replication model?
A
- the second replication results in 4 double-stranded helices
- two with OG parental and new strand
- two helices with completely new strands
21
Q
What is the conservative replication model?
A
- model considered in late 50ies
- predicted that during first replication the parental helix would be fully conserved and the new strands would combine to form a completely new helix
- the second replication would then result in one completely conserved parental helix and three completely new helices
22
Q
Who proved that the semi-conservative model of replication was the correct one?
A
- Matthew Meselson and Franklin Stahl
23
Q
What technique was used to prove that the semi-conservative model of replication was the correct one?
A
- Meselson and Stahl used a centrifuge tube that was filled with a heavy salt sol’n and DNA fragments
- this was spun for several days
- density gradient developed within tubes with heavy DNA at the bottom of the tube
called: CESIUM CHLORIDE (CsCl) EQUILIBRIUM-DENSITY GRADIENT CENTRIFUGATION
24
Q
How does CsCl equilibirum-density gradient centrifugation work?
A
- CsCl separates double-stranded DNA (dsDNA) molecules of different densities
- heavier DNA sediments further down CsCl gradient and lighter DNA migrates near top
- newly formed DNA strands are labeled with a form of nitrogen (14N) that is lighter
- parental is just has the natural 15N, which is more dense
25
What organism did Meselson and Stahl use and why did results confirm semi-conservative?
- used e. coli bacterium
- semi-conservative because after the first replication there was only a single band with one intermediate weight (between N15 and 14), meaning all had the same form - could have been semi-conservative or dispersive
- but after second round there where two bands, one intermediate one and one light one, meaning that now we had two types of helices where dispersive model would have resulted in only one type: a hybrid
26
What kind of replication occurs in most circular DNA?
Theta (θ) replication
| - occurs for instance in E.coli and in certain bacterial plasmids
27
What are the steps of Theta Replication?
1. Double stranded DNA unwinds at replication origin
2. single-stranded template for synthesis of new DNA is produced at origin and replication bubble can form
3. the replication forks at each end proceed to go around the circle
4. Eventually, two circular DNA molecules are produced
28
In which direction does Theta Replication proceed?
bidirectional
29
What are the products of Theta Replication?
two circular DNA molecules
30
What is the definition of Theta Replication?
= utilizes semi-conservative replication of a circular double helical DNA to produce two double stranded helices through an intermediate that resembles the Greek letter theta
31
What kind of replication occurs in the F factor and in some viruses?
Rolling Circle Replication (specialized form of replication)
| - used during transformation of F factor DNA in E.coli
32
What are the steps of rolling circle replication?
1. Replication is initiated by break in one of the nucleotide strands
2. DNA synthesis begins at 3' end of the broken strand and the inner strand is the template, the 5' end of broken strand is displaced
3. Cleavage releases single stranded linear DNA and double-stranded circular DNA
4. The linear DNA may circularize and serve as a template for synthesis of a complementary strand
33
In what direction that rolling circle replication occur?
unidirectional beginning at 3' end of the broken stand
34
What are the products of rolling circle replication?
= multiple circular DNA molecules
35
What kind of replication occurs in linear chromosomes?
= linear chromosome replication
| - occurs in eukaryotic cells
36
What are the steps of linear chromosome replication?
1. Each chromosome contains numerous origins and at each origin the DNA unwinds and produces a replication bubble
2. DNA synthesis takes place on both strands at each end of the bubble as the replication forks proceed outward
3. Eventually, the forks of adjacent bubbles run into each other and the segments of DNA fuse
4. when the segments fuse, two identical linear DNA molecules are produced
37
In which direction does linear chromosome replication proceed?
= bidirectional
38
What are the products of linear chromosome replication?
= two linear DNA molecules (semi-conservative) that are identical chromosomes
39
What are the key requirements of DNA replication?
1. Magnesium
2. DNA dependent DNA polymerase
3. Four deoxyribonucleoside triphosphates (dNTPs)
4. a template strand to be copied
5. an RNA primer that provides the 3'-OH end to initiate DNA synthesis by DNA polymerase
40
How is the triphosphate group in the four dNTPs used in DNA replication?
- only the alpha phosphate (the first one after the base) is incorporated into the DNA strand
- the beta and gamma phosphates are eliminated from each dNTP during the base incorporation step
41
Which end of the newly synthesized DNA strand is the growing end?
= 3' OH is the growing end of DNA
42
In which direction is DNA always synthesized and by what?
- in the 5' to 3' direction using the 3' to 5' parental strand as the template (the 3' OH group of the last nucleotide on the strand attacks the 5' phosphate group of the incoming dNTP)
- synthesized by DNA polymerase
43
Where do phosphodiester bonds form during DNA synthesis?
- between the 5' alpha phosphate of the incoming dNTP and the 3' OH of the nucleotide in the growing DNA strand (beta and gamma phosphates are cleaved during incorporation)
44
What are key features of DNA replication?
- DNA is always synthesized in the 5' to 3' direction
- newly synthesized DNA strand is complementary and anti-parallel to the parent strand
- DNA strands are held together by hydrogen bonds between complementary bases
45
What is new DNA synthesized from?
- from deoxyribonucleic triphosphates (dNTPs)
46
A fragment of partially double-stranded DNA has the structure
5' AGCTAGTTATTACG 3'
TCAATAAT
If this DNA was used as a template for replication, which nucleotide would be incorporated first?
- since DNA strands are antiparallel the bottom strand must be 3' on the left side and 5' on the right side
- therefore the first nucleotide to be incorporated would be:
5' AGCTAGTTATTACG 3'
XTCAATAAT
An A where the X is
47
What does nuclease do to DNA?
- both single and double-stranded DNA is cleaved by nuclease
- Nucleases cleave the phosphodiester backbone and leave the alpha phosphate group attached to either the 5' carbon of their original nucleotide or the 3' hydroxyl of the preceding 5' nucleotide
- depending on where nuclease cuts, phosphate will reside in the original nucleotide or the adjacent 5' nucleotide of each chain
48
Describe DNA synthesis on the leading strand:
- on the leading strand DNA synthesis is continuous
- 5' to 3' means the leading strand grows as the strand unwinds
- results in one continuous strand since it can continuously add to the 3' end of the synthesized strand
- leading strand always proceeds in the same direction as unwinding
49
Describe DNA synthesis on the lagging strand:
- on the lagging strand DNA synthesis is discontinuous
- synthesis begins at the fork and runs from the fork to the end of the template strand, meaning it runs out of template
- each time it does a new fragment is created from the fork to where the previous strand started, resulting in small fragments of DNA produced by discontinuous synthesis (but is also always 5' to 3' growing)
- always proceeds in direction opposite to unwinding
50
What are Okazaki fragments?
= the short DNA fragments produced by discontinuous DNA synthesis on the lagging strand
- since they are discontinuous each okazaki fragment must start with a new primer
51
Where does all DNA synthesis start?
- ALL DNA synthesis begins at an origin of replication and proceeds bidirectionally from this point of unwinding
- in circular and linear chromosomes this means two replication forks, each with a leading and lagging strand synthesis, are created
52
What is the replication origin called in E. coli?
= oriC
- occupies a 245 bp segment and consists of 4 nine base pair sequences that bind a protein called DnaA
- when DnaA binds, an adjacent AT rich region unwinds and creates a region of single-stranded DNA that permits leading and lagging strand DNA synthesis to begin
- the unwinding allows helicase and other single-stranded-binding proteins to attach at single-strand
53
What is the role of single-stranded binding proteins in DNA synthesis in prokaryotes?
- they keep the DNA single-stranded and linear which assists in the recruitment of DNA helicase to each side of the open area to begin further unwinding of the double-stranded DNA
- they coat the strand and stabilize it
54
What are key features of DNA helicase in prokaryotic DNA replication?
- DNA helicase unwinds the DNA in 5' to 3' direction travelling on the side of the lagging strand ahead of replication machinery
- it breaks hydrogen bonds and moves the replication fork
55
What is the function of topoisomerase II (DNA Gyrase) in prokaryotic DNA replication?
- helix unwinding creates stress ahead of where the unwinding is occurring
- if the stress is not relieved, the helicase will not be able to continue unwinding the double-stranded DNA
- topoisomerase II proceeds along the DNA ahead of where the helicase unwinds the DNA and relieves the tension through nicking the DNA strand
56
What does the helicase-induced unwinding of double helical DNA cause ahead of helicase?
- causes the strands to be overwound, which produces positive supercoils that could stop replication
- in bacteria topoisomerase II nick the DNA to release the positive supercoils (DNA wraps over itself many times)
- DNA gyrase is on either side of the replication bubble and releases tension as well as resealing the strands
57
What is the role of DNA primase?
- primase is recruited to the replication fork and synthesizes a short RNA primer
- the RNA primer provides the 3' OH end that permits the leading strand replication to proceed
- as helicase unwinding proceeds the DNA primase adds a primer to begin 5' to 4' lagging strand DNA syntehsis
- this allows bidirectional replication to occur away from the replication since a primer is needed so DNA polymerase can add nucleotides to the 3' end
58
What are the primers in DNA synthesis?
= a short stretch of RNA nucleotides that provide a 3'OH group to which DNA polymerase can add DNA nucleotides
59
How many DNA polymerases are there in E.coli?
- 5
- I and III are used in chromosomal DNA replication = replicative polymerases
- II, IV, V are used for DNA repair functions
60
What are key facts about DNA Polymerase I?
- aids in removal of RNA primers = converts Okazaki fragments into continuous piece of DNA when it replaces primers with short tract of DNA
- has 5' to 3' polymerase and 5' to 3' exonuclease activity, which assists in removing RNA primers
- 3' to 5' proofreading exonuclease activity
- not highly processive; short tract synthesis
61
What are key facts about Polymerase III?
- has 5' to 3' polymerase activity
- lacks 5' to 3' exonuclease activity
- has 3' to 5' proofreading exonuclease activity
= main replicative polymerase and can copy long stretches of DNA (i.e. highly processive)
62
What is the beta sliding clamp?
= a ring-shaped polypeptide (protein) that encircles the DNA and interacts with DNA polymerase III to enhance processive DNA synthesis
- it helps to keep the polymerase on the DNA and promotes processive (continuous) DNA syntehsis
63
What permits unrestricted DNA replication on both the leading and lagging strand?
- the combination of POL III, the beta sliding clamp, and single-stranded-binding protein SSB that keeps the DNA free of secondary structures
64
Describe how the lagging strand is converted into a continuous DNA strand:
- when DNA synthesis by Pol. III reaches the 5' end of the RNA primer, Pol III is swapped for Pol I
- DNA polymerase I removes the RNA primer and re-synthesizes a short tract of DNA
- DNA ligase makes a phosphodiester bond b/n 5' phosphate and the 3' OH group which seals the nick and creates continuous strand
65
Why can't Pol III remove the RNA primer?
- b/c it does not have a 5' to 3' exonuclease activity
66
What has to happen in order for both lagging and leading strand to be replicated simultaneously?
- the DNA must form a loop because the polymerase must release the template and shift to a new position further along template to resume synthesis of the lagging strand
67
Where in the machinery of replication is DNA helicase located?
on the lagging strand
68
What is the polarity of DNA strands?
- 5' to 3'
69
Summary: what all is needed for replication mechanism?
- topoisomerase
- helicase
- single strand DNA binding protein (SSB)
- DNA primase
- DNA polymerase III (plus beta clamp)
- DNA polymerase I
- DNA ligase
70
What is the typical error frequency in replicative polymerase?
- 1 error in every 10^10 nucleotides added (inherent polymerase accuracy plus cellular mutation repair system)
71
What are unique aspects of eukaryotic chromosome replication?
- shorter RNA primers and okazaki fragments
- DNA replication occurs only during S phase
- multiple polymerases (at least 15)
- bidirectional replication from multiple origins of replication on each chromosome
- nucleosomes (all 9 histone proteins) have to be removed from parental DNA and properly reassembled on newly synthesized DNA
- telomeres shorten at each round of eukaryotic replication
72
What are the 3 most important polymerases in eukaryotic chromosome replication and their functions?
1) Pol Epsilon - performs leading strand replication
2) Pol Delta - performs lagging strand replication
3) Pol Alpha - has primase activity
73
How do linear chromosomes prepare for replication and when does it occur?
- prepare eukaryotic origins for replication in the G1 phase (origin licensing: involves assembly and phosphorylation of origin specific replication proteins)
- replication begins in S phase
74
What happens in G1 phase?
Growth and metabolism
| - preparation of origins for replication
75
What happens in S phase?
- DNA synthesis and chromosome duplication
76
What happens in G2 phase?
- Preparation for mitosis
77
What is Interphase?
- consists of G1, S phase, and G2
| - interrupted by division (M phase)
78
What is the telomere problem?
= when replication machineray reaches the telomere at both chromosome ends there is a loss of information due to the removal of the RNA primer on the lagging strand (degradation of chromosome ends)
- essentially, there is a gap left at the end of each chromsome
- if this occurs at each round of replication, chromosome shortening will occur
- this is unique to linear eukaryotic chromosomes
79
What does Telomerase activity do?
- extends eukaryotic chromosome ends in replicating cells and circumvents shortening of chromosomes
- telomerase is an enzyme
- has an RNA component whose sequence is complementary to the TTAGGG repeats found in telomeres, when it binds to chromosome ends through base paring, RNA templated DNA synthetic activity can extend the length of the chromsome end in 5' to 3' direction
- telomerase dissociates and then RNA primer is synthesized and complementary DNA strand is synthesized by DNA polymerase
- the complementary activity of these enzymes keeps telomeres from shortening
80
What are short telomeres associated with?
- cellular senescence (cell cycle arrest)
- premature aging (Progeria disease and Werner's syndrome)
- cell death
81
Telomerase activity in somatic cells vs cancerous cells?
- low telomerase activity in somatic cells
| - high telomerase activity in cancer cells (promotes growth)
MBG 2040
flashcards
Decks in class (9)
# Cards
