lecture 2 (chapter 11) dna replication 1. “it has not escaped our notice that the specific pairing...
TRANSCRIPT
LECTURE 2
(Chapter 11)
DNA Replication
1
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
-James Watson and Francis Crick (1953)
2
INTRODUCTION
• DNA replication is the process by which the genetic material is copied– The original DNA strands are
used as templates for the synthesis of new strands
– Each strand carries the information for making the other strand
3
11.1 STRUCTURAL OVERVIEW OF DNA REPLICATION
• DNA replication relies on the complementarity of DNA strands– The AT/GC rule or Chargaff’s rule
4
• Complement: (Noun) A thing that completes or brings to perfection
• Conflate: (Verb) Combine (two or more texts, ideas, etc.) into one.
• Virulent: (Adj) Able to overcome bodily defensive mechanisms: markedly pathogenic
• Lyse: (Verb) To cause dissolution or destruction of cells• Conservative: (Adj) Disposed to observe existing
conditions
5
WORDS TO KNOW…
• The two complementary DNA strands come apart
• Each serves as a template strand for the synthesis of a new complementary DNA strand– The two newly-made DNA strands =
daughter strands– The two original DNA strands = parent
strands
6
7
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T A
G
C
A
G
A T
T A
T
G
GA
A
C
C
CT
T
G
C G
T A
T A
C
G
A T
T A
C G
T
A T
C G
C
C G
A T
C G
C A
CGG
C
Incomingnucleotides
Original(template)strand
Original(template)strand
Newlysynthesizeddaughter strand
Replicationfork
(a) The mechanism of DNA replication (b) The products of replication
Leadingstrand
Laggingstrand
5′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
5′ 3′
5′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
5’′ 3′
3′ 5′
A T
A T
T A
T A
T A
C G
C G
G CG C
G CG C
C G
A T
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′3′
A
A T
C
Figure 11.1
Identical base sequences
A pairs with T and G pairs with C during synthesis of a new
strand
8
Meselson Stahl Experiment (1958)
9
• Meselson and Stahl sought to distinguish between three hypothetical models for the replication of DNA
• In the late 1950s, three different mechanisms were proposed for the replication of DNA– Conservative model
• Both parental strands stay together after DNA replication
– Semi-conservative model• The double-stranded DNA contains one parental and one
daughter strand following replication• Now known to be correct
– Dispersive model• Parental and daughter DNA are interspersed in both strands
following replication
10
Figure 11.2
(a) Conservative model
First round ofreplication
Second roundof replication
Originaldoublehelix
(b) Semiconservative model (c) Dispersive model
11
The Hypothesis
– Based on Watson’s and Crick’s ideas, the hypothesis was that DNA replication is semi-conservative.
– Started by growing a E. coli cells in heavy nitrogen (15N)for many generations
• This ensured that all the nitrogen atoms in the E. coli cells was heavy
• “Heavy” nitrogen has more mass than normal nitrogen (14N).
12
Figure 11.3
Experimental level Conceptual level
2. Incubate the cells for various lengths oftime. Note: The 15N-labeled DNA isshown in purple and the 14N-labeledDNA is shown in blue.
3. Lyse the cells by the addition oflysozyme and detergent, whichdisrupt the bacterial cell wall
andcell membrane, respectively.
4. Load a sample of the lysate onto a CsCl gradient containing ethidium bromide. (Note: The average density of DNA is around 1.7 g/cm3, which is well isolated from other cellular
macromolecules.)
5. Centrifuge the gradients until the DNAmolecules reach their equilibriumdensities.
6. DNA within the gradient can beobserved under a UV light.
DNA
Cell wall
Cell membrane
LightDNA
Half-heavyDNA
HeavyDNA
UVlight
(Result shown here isafter 2 generations.)
CsClgradient
Lysate
Lysecells
37°C
14Nsolution
Suspension ofbacterialcells labeledwith 15N
Up to 4 generations
Density centrifugation
Generation
0
1
Add 14N
2
1. Add an excess of 14N-containingcompounds to the bacterial cells soall of the newly made DNA will
contain14N.
13
14
15
Light
Half-heavy
Heavy
Generations After 14N Addition
4.1 3.0 2.5 1.9 1.5 1.1 1.0 0.7 0.3
*Data from: Meselson, M. and Stahl, F.W. (1958) The Replication of DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 44: 671−682
Interpreting the Data
After one generation, DNA is “half-heavy”
This is consistent with both semi-conservative and dispersive
models
After ~ two generations, DNA is of two types: “light” and “half-
heavy”This is consistent with only the semi-conservative
model16
Reverse image, UV light
11.2 DNA REPLICATION IN BACTERIA
• Figure 11.4 presents an overview of the process of bacterial chromosomal replication
– DNA synthesis begins at a site termed the origin of replication
• Each bacterial chromosome has only one origin of replication
– Synthesis of DNA proceeds bidirectionally around the bacterial chromosome
– The two replication forks eventually meet at the opposite side of the bacterial chromosome
• This ends replication
17
0.25 μm
(b) Autoradiograph of an E. coli chromosome in the act of replication
(a) Bacterial chromosome replication
Replicationforks
Origin ofreplication
Replicationfork
Site wherereplicationends
From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press.
Replicationfork
Figure 11.4
18
Catanenes
– Nucleic acids (in order of appearance):• Origin of replication (Ori C)• Template strands• Free ribonucleotide triphosphates (NTPs)• RNA primers• Free deoxyribonucleotide triphosphates dNTPs)• Daughter strands
19
The Players
– Proteins (in order of appearance):• DnaA proteins• DNA helicase• Single-stranded DNA binding proteins• DNA gyrase (topoisomerase)• DNA primase• DNA polymerase III• DNA polymerase I• Ligase
20
The origin of replication in E. coli is termed oriC Origin of Chromosomal replication It is comprised of DNA Both chromosomal DNA and plasmids have one
Three types of DNA sequences in oriC AT-rich region DnaA boxes GATC methylation sites
Refer to Figure 11.5
Initiation of Replication
21
Figure 11.5
E. colichromosome
oriC
G GG G GGGA GAGAAAAAA GAA AAT
T
T T ATT TTTA ATTTTTC T TC ATTCT TCCC
1
CC C CCCT CTCTTTTTT CTT TTA A A T AA AAAT TAAAAAG A AG T AAGA AGG
T AG T CCTT AACAAGGAT AGC CAG T T CCT T
T
CGDnaA box
DnaA box
DnaA box
DnaA box
DnaA box
T TGGATCA T CG CTGGA GGA TC A GGAA TTGTTCCT A TCG GTC A A GGA AGCA ACCTAGT A GC GACCT CCA
T CT ACAT GAATCCTGG GAA GCA A A ATT GGAA TCTGAAA A CT ATGTG TA
A
G
C CC C GGTT TACAGCTGG CT
T
T
ATG A A TGA TCGG AGTTACG G AA AAAAC GAAG GG G CCAA ATGTCGACC GT A TAC T T ACT AGCC TCAATGC C TT TTTTG CTT
A GC A TACT GA CGTTCT GTG AGG G T CTA CTCC TGGTTCA T AA CTCTC AAAT CG T ATGA CT AGCAAGA ACCTCC C A GAT GAGG ACCAAGT A TT GAGAG TTT
GA T GTAC CAGTA CA GCA T CAGG CACT A CATG GTCAT GT A CGT A GTCC GT
A GA A TGTA CTT AGGACC CTT CGT T T T AA CCTT AGACTTT T GA T ACAC ATC
AT-rich region
5′–
–
50
51 100
101 150
201
251 275
250
151 200
3′
22
AT-rich region DnaA boxes
Next, DNA helicases bind
DnaA protein
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
5′ 3′
AT- rich
region
3′ 5′
5′3′ 3′
5′
Figure 11.6
DNA replication is initiated by the binding of DnaA proteins to the DnaA box sequences
This binding stimulates the cooperative binding of additional ATP-bound DnaA proteins to form a large complex
23
Figure 11.6Composed of six subunits
Travels along the DNA in the 5’ to 3’ direction
Uses energy from ATP
Bidirectional replication is initiated
Helicase
DNA helicase separates the DNA in bothdirections, creating 2 replication forks.
ForkFork
5′3′
5′3′
3′5′
3′
5′
24
DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead
of each replication DNA gyrase (AKA topoisomerase) travels
ahead of the helicase and alleviates the supercoiling http://www.youtube.com/watch?
v=EYGrElVyHnU
25
Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase DNA primase is a specialized type of RNA
polymerase DNA primase “reads” the template strand and
synthesizes short complementary RNA strands called “primers”
The RNA primers “prime” the DNA for being copied by DNA polymerase
DNA polymerases cannot synthesize DNA unless the template strands have been “primed” by primase
26
• Prime: (Verb) Make something ready for use or action.
• RNA Polymerase: (Noun) An enzyme that reads single-stranded DNA and makes an RNA copy of it.
• DNA Polymerase: (Noun) An enzyme that reads single-stranded DNA and makes a DNA copy of it.
• Fidelity: (Noun): (1) The state or quality of being faithful; (2) Accuracy in details: Exactness
• Processive: (Adj) Ability to advance or go forward
27
MORE WORDS TO KNOW…
Next, as each fork opens, DNA polymerase III extends the primers from their 3’ ends to make daughter strands
28
Problem is overcome by the RNA primers synthesized by
primase
DNA polymerases cannot initiate DNA synthesis
Able tocovalently linktogether
Unable tocovalently linkthe 2 individualnucleotides together
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Primer
DNA polymerases are the enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand
In E. coli there are five proteins with polymerase activity DNA pol I, II, III, IV and V DNA pol I and III
Normal replication DNA pol II, IV and V
DNA repair and replication of damaged DNA
DNA Polymerases
29
DNA pol I First DNA polymerase discovered (Kornberg) Composed of a single polypeptide Removes the RNA primers and replaces them with DNA
DNA pol III Responsible for most of the DNA replication Composed of 10 different subunits
The a subunit catalyzes bond formation between adjacent nucleotides (DNA synthesis)
The other 9 fulfill other functions The complex of all 10 subunits is referred to as the DNA
pol III holoenzyme
DNA Polymerases
30
(a) Schematic side view of DNA polymerase III
3′
3′ exonucleasesite3′
5′ 5′
Fingers
Thumb
DNA polymerasecatalytic site
Templatestrand
Palm
Incomingdeoxyribonucleoside triphosphates(dNTPs)
Bacterial DNA polymerases may vary in their subunit composition However, they all have the same type of catalytic subunit
Figure 11.8
Structure resembles a human right hand
Template DNA is threaded through
the palm;
Thumb and fingers wrapped around the
DNA
31
The two new daughter strands are synthesized in different ways Leading strand
One RNA primer is made at the origin DNA pol III attaches nucleotides in a 5’ to 3’ direction
as it slides toward the opening of the replication fork
Lagging strand Synthesis is also in the 5’ to 3’ direction
However it occurs away from the replication fork Many RNA primers are required DNA pol III uses the RNA primers to synthesize small
DNA fragments (1000 to 2000 nucleotides each) These are termed Okazaki fragments after their discoverers
32
DNA polymerases can attach nucleotides only in the 5’ to 3’
direction
Problem is overcome by synthesizing the new strands both toward, and away from, the
replication fork
(b)
(a)
3′
5′
5′
3′
3′
5′
5′
3′Cannot link nucleotides in this direction
Can link nucleotidesin this direction
33
DNA pol I removes the RNA primers and fills the resulting gap with DNA It uses a 5’ to 3’ exonuclease activity to digest the RNA
and 5’ to 3’ polymerase activity to replace it with DNA After the gap is filled a covalent bond is still
missing DNA ligase catalyzes the formation of a
phosphodiester bond Thereby connecting the DNA fragments
34
Figure 11.10
Origin of replication
Replicationforks
Direction ofreplication fork
FirstOkazakifragment
First and second Okazakifragments have beenconnected to each other.
First Okazaki fragmentof the lagging strand
SecondOkazakifragment
ThirdOkazakifragment
Primer
Primer
The leading strand elongates,and a second Okazaki fragmentis made.
The leading strand continues toelongate. A third Okazakifragment is made, and the firstand second are connectedtogether.
Primers are needed to initiate DNA synthesis.The synthesis of the leading strand occurs inthe same direction as the movement of thereplication fork. The first Okazakifragment of the lagging strand ismade in the opposite direction.
5′
5′
5′
5′
3′
5′
3′
3′
5′
3′
3′
3′
5′
5′
5′
5′
3′
3′
3′
3′
5′
5′3′
3′
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Leadingstrand
DNA strands separate atorigin, creating 2 replicationforks.
35
5′
3′ 5′
3′
Origin of replication
Replication fork Replication fork
Leadingstrand
Laggingstrand
Leadingstrand
Laggingstrand
Figure 11.11
The synthesis of leading and lagging strands from a single origin of replication
36
"For example the statement i = 1/7;will assign the value zero to the integer variable i. Note that if the quotient of two integers is assigned to a float then the same loss of accuracy still occurs. Even if i in the above assignment was a variable of type float 1/7 would still be evaluated as an integer divided by an integer giving zero, which would then be converted to the equivalent float value, i.e. 0.0, before being assigned to the float variable i."
37
Quote from my son’s computer science textbook:
38
Equivalent quote from my Dr. Ballard’s lecture on DNA replication:
"Lagging strand synthesis begins when DNA primase lays down an RNA primer in the 5' to 3' direction on the strand where doing so places the 3' end of the primer farther from the nearest fork than the 5' end. As the fork opens and additional DNA becomes exposed, another primer is laid down, again in the 5' to 3' direction. In this way, as the fork opens, the DNA is made 5' to 3' in multiple Okazaki fragments connected by short areas of RNA. DNA polymerase I removes the RNA and replaces it with DNA, after which the phosophodiester backbone between the Okazaki fragments is healed with DNA ligase..."
Which is why animations are essential…
39
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40
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DNA polymerases catalyzes the formation of a covalent (ester) bond between the Innermost phosphate group of the incoming
deoxyribonucleoside triphosphate AND
3’-OH of the sugar of the previous deoxynucleotide In the process, the last two phosphates of the
incoming nucleotide are released In the form of pyrophosphate (PPi) Refer to figure 11.12
The Reaction of DNA Polymerase
41
Figure 11.12
O
OOO
O
O
P CH2
O2
OOO P CH2
O2
OOO
CH2
O2
O
OO
O2 P
O2
5′
OOO
O
O
P CH2
OO P CH2
O2
O
OOO P CH2
O2O
O P
O2
O
P
O2
O2O2 +
OH
OH
OH
PP
O
O
OO
O
O
PH2C
H2C
O2
O
OO P
O
O
H2C
O2
OO P
O2
O
OO
O
O
PH2C
H2C
O2
O
OO P
O
O
H2C
O2
OO P
O2
O
O2
O2
New DNA strand Original DNA strandCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
O
OOO
O
O
P CH2
O –
Incoming nucleotide(a deoxyribonucleosidetriphosphate)
Pyrophosphate (PPi)
Newesterbond
5′
5′ end
OOO P CH2
O –
OOO
CH2
O –
O
OO
O – P
O –
5′
OOO
O
O
P CH2
5′
5′ end
3′ end
OO P CH2
O –
O
OOO P CH2
O –O
O P
O –
O
P
O –
O –O– +
OH
3′
OH
3′
OH
3′
PP
O
Cytosine Guanine
Guanine Cytosine
ThymineAdenine
Cytosine Guanine
Guanine Cytosine
Thymine Adenine
O
OO
O
O
PH2C
H2C
O –
3′
O
OO P
O
O
H2C
O –
OO P
O –
5′
5′ end
O
OO
O
O
PH2C
H2C
O –
3′
O
OO P
O
O
H2C
O –
OO P
O –
5′
3′ end
5′ end
O
O –
O –
3′ end
Innermost phosphate
42
DNA polymerase III remains attached to the template as it is synthesizing the daughter strand
This processive feature is due to several different subunits in the DNA pol III holoenzyme b subunit forms a dimer in the shape of a ring around
template DNA It is termed the clamp protein Once bound, the b subunits can freely slide along dsDNA Promotes association of holoenzyme with DNA
g complex catalyzes b dimer clamping to the DNA It is termed the clamp-loader complex Includes , , g d d’, c and y subunits
DNA Polymerase III is a Processive Enzyme
43
The effect of processivity is quite remarkable
In the absence of the b subunit DNA pol III falls off the DNA template after about 10
nucleotides have been polymerized Its rate is ~ 20 nucleotides per second
In the presence of the b subunit DNA pol III stays on the DNA template long enough to
polymerize up to 500,000 nucleotides Its rate is ~ 750 nucleotides per second
DNA Polymerase III is a Processive Enzyme
44
On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences These are designated T1 and T2
T1 stops counterclockwise forks, T2 stops clockwise forks
The protein tus (termination utilization substance) binds to the ter sequences tus bound to the ter sequences stops the movement of
the replication forks
Refer to Figure 11.13
Termination of Replication
45
Fork
Fork
Fork
Fork
ter(T2)
oriC
oriC
(T1)
Tus
Tus
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
ter
Figure 11.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Prevents advancement of fork moving right-to-left (counterclockwise fork)
Prevents advancement of fork moving left-to-right (clockwise fork)
46
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DNA replication ends when oppositely advancing forks meet (usually at T1 or T2)
Finally DNA ligase covalently links the two daughter strands
DNA replication often results in two intertwined molecules Intertwined circular molecules are termed catenanes These are separated by the action of topoisomerase
Termination of Replication
11-3647
11-37
Figure 11.14
Catenanes
Catalyzed by DNA topoisomerase
Replication
Decatenation viatopoisomerase
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48
DNA replication exhibits a high degree of fidelity Mistakes during the process are extremely rare
DNA pol III makes only one mistake per 108 bases made
There are three main reasons why fidelity is high 1. Mismatched pairs don’t hydrogen bond properly and
are therefore unstable Mistake is corrected before the nucleotide is added Error rate for mismatched base pairs is 1 per 1,000 nucleotides
2. The DNA polymerase active site only accommodates properly matched bases Mistake is corrected before the nucleotide is added “Induced-fit” phenomenon decreases the error rate to a range of
1 in 100,000 to 1 million
Replication Fidelity
49
3. DNA polymerase can “proofread” its mistakes immediately after they happen and remove them
DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand
The enzyme uses a 3’ to 5’ exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed
DNA synthesis then resumes in the 5’ to 3’ direction Refer to figure 11.16
Added to first two mechanisms, lowers error rate to 1 in 108 to 1 in 109
Proofreading
50
A schematic drawing of proofreadingFigure 11.16
Site where DNA backbone is cut
C
T
3′
3′
5′ 5′
Mismatch causes DNA polymerase to pause,leaving mismatched nucleotide near the 3′ end.
Templatestrand
The 3′ end enters theexonuclease site.
3′
5′ 5′
At the 3′ exonuclease site,the strand is digested inthe 3′ to 5′ direction until theincorrect nucleotide isremoved.
3′
5′ 5′
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Base pair mismatch near the 3′ end
3′
3′
Incorrectnucleotideremoved
exonucleasesite
51
52
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Assuming…
• The average size of a human gene is ~20,000 bp• The rate of error by DNA polymerase is 10-9 mutations/bp
Then…• The chance of an average human gene mutating in any replication
cycle can be calculated as
(10-9 mutations/bp)(20,000 bp/gene) = 0.00002 mutations/gene
So there is only a 1 in 50,000 chance that a gene will suffer a mutation in any given replication cycle!
Example of how this plays out(back of the envelope calculation)
53
Kornberg’s Experiments (1950s)
• First person to isolate DNA polymerase and study its activity in vitro– Classic biochemist!
• He received a Nobel Prize for his efforts in 1959
54
55
• Experimental tools:– Extract of E. coli proteins– 32P-labled deoxynucleotide triphosphates– Template DNA (not radioactively labeled)– Weak solution of perchloric acid– Centrifuge– Water bath– Scintillation counter (bee-beep!)
• Kornberg hypothesized that deoxyribonucleoside triphosphates (dNTPs) are the precursors of DNA synthesis
• Developed an assay to test for the presence of an enzyme that could make DNA from dNTPs
• His experimental hook:– Long DNA strands can be precipitated in a weak
acidic solution while free nucleotides cannot
56
• In this experiment, Kornberg mixed the following– An extract of proteins from E. coli– Template DNA– Radiolabeled nucleotides
• These were incubated for sufficient time to allow the synthesis of new DNA strands– Addition of acid will precipitate these DNA strands– Centrifugation will separate them from the
radioactive nucleotides leftover in the supernatant (unincorporated)
57
The Hypothesis
– DNA synthesis can occur in vitro if all the necessary components are present
– Therefore, an in vitro system can be developed to purify and study the enzyme that makes DNA
Testing the Hypothesis
Refer to Figure 11.19
58
Figure 11.19
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Experimental level
Add perchloric acid.
Conceptual level
Mix together the extract of E. coliproteins, template DNA that is notradiolabeled, and 32P-radiolabeleddeoxyribonucleoside triphosphates. Thisis expected to be a complete system thatcontains everything necessary for DNAsynthesis. As a control, a second sampleis made in which the template DNA wasomitted from the mixture.
1.
Incubate the mixture for 30 minutes at 37°C.
2.
Add perchloric acid to precipitate DNA. (It does not precipitate free nucleotides.)
3.
Centrifuge the tube. Note: The radiolabeled deoxyribonucleosidetriphosphates that have notbeen incorporated into DNA will remainin the supernatant.
4.
Collect the pellet, which contains precipitated DNA and proteins. (Thecontrol pellet is not expected tocontain DNA.)
5.
Count the amount of radioactivity in the pellet using a scintillation counter.(See the Appendix.)
6.
37°C 37°C
Template DNA
Complete system
Control
Supernatant
Pellets
Template DNA
E. coli proteins
E. coli proteins
32P-labeled deoxyribonucleosidetriphosphates (dNTPs)
32P-radiolabeled deoxyribonucleosidetriphosphates
Free labeled nucleotides
DNA with 32P-labelednucleotides in new strand
32P32P
32P32P
32P
32P32P
32P32P
32P
32P32P
32P32P
32P32P 32P
32P32P
32P
32P
G
G G
G
T
T
A
AA A
C
C
C C
C
C
AA
T
T
G
G
CC
TT
A
A
C
CG
G
C G A T CT TG C T A GA A
59
Interpreting the Data
E. coli proteins + nonlabeled template DNA +
radiolabled nucleotides
= Radiolabeled product
Expected result because the template is necessary for
replication
Taken together, these results indicate that this technique can be used to detect and measure the synthesis of DNA in vitro
60
• Once he had the assay working with the complete protein extract he began testing fractions of the extract– Column chromatography– Systematic separation exploiting charge,
hydrophobicity, size, and other characteristics– Each time a protein fraction tested positive for DNA
polymerase activity, he retained it and continued fractionating it
– Ultimately obtained a “pure” extract of DNA polymerase I
• Initially called the Kornberg Enzyme
61
62
11.3 EUKARYOTIC DNA REPLICATION
• Eukaryotic DNA replication is not as well understood as bacterial replication
– The two processes do have extensive similarities,• The types of bacterial enzymes described in Table 11.1 have
also been found in eukaryotes
– Nevertheless, DNA replication in eukaryotes is more complex
• Many origins of replication per chromosome• Chromosomes are linear rather than circular
63
Eukaryotes have long linear chromosomes They therefore require multiple origins of replication
To ensure that the DNA can be replicated in a reasonable amount of time
DNA replication proceeds bidirectionally from many origins of replication Refer to Figure 11.22
Multiple Origins of Replication
64
Figure 11.22
Chromosome Sister chromatids
Before S phase During S phase End of S phase
Origin
Origin
Origin
Origin
Origin
Centromere
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65
Linear eukaryotic chromosomes have telomeres at both ends
The term telomere refers to the complex of DNA sequences and bound proteins
Linear Chromosomes, Death, and Cancer
66
Telomeres protect the free ends of linear chromosomes from Recombination with other free ends Damage and degradation
Bacterial chromosomes do not need this protection Their chromosomes are circular; do not have ends
Linear Chromosomes, Death, and Cancer
67
Telomeric sequences consist of Moderately repetitive tandem arrays 3’ overhang that is 12-16 nucleotides long
Figure 11.24
Telomeric sequences typically consist of Several guanine nucleotides Many thymine nucleotides
Refer to Table 11.5
Telomeric repeat sequences
OverhangCG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
AT
CG
CG
CG
TA
AT
TAT G G GA
AT
AT G G GAT AT T T GGG
5′
3′
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As we’ve already seen, DNA polymerases possess two unusual limitations 1. They synthesize DNA only in the 5’ to 3’ direction 2. They cannot initiate DNA synthesis
These two features pose a problem at the 3’ ends of linear chromosomes-the end of the strand cannot be replicated! This is known as the “end replication problem” Bacterial chromosomes do not have this problem
because they are circular
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We’ll demonstrate this on the board
The end replication problem causes eukaryotic chromosomes to get progressively shorter each time the DNA (and cell) divides
How do eukaryotic cells cope with this? Single-celled eukaryotes (e.g. yeast) produce an
enzyme called telomerase that heals the damage Therefore, their chromosomes are healed before
they are passed to the next generation
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Death phase is due to running out of food
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Log
of
nu
mb
er
of
hu
man
liv
er
cells in
cu
ltu
re NO RECOVERY
Even if fed, death phase will occur due to telomere damage
Multi-cellular organisms (like us) make telomerase but express it primarily in germ-line tissue This ensures that telomeres do not shorten with the
generations Expression of telomerase is much lower in
somatic tissues Explains why cultured cells are not immortal Also explains (in part) why multi-celled organisms
have a limited life span
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Humans have trillions of cells Lots of cell division = lots of opportunity for
mutations If mutations affect cell cycle control genes, result is a
tumor If telomerase is active in the cells in the tumor, it
becomes cancerous Therefore, telomerase is kept “off” in somatic
tissues Trade-off for no cancer is a limited life span!
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Sample Problem(more than one answer may be correct)
Which of the following express high levels of telomerase?
a. Yeast cells
b. E. coli cells
c. Human breast tissue
d. Human testicular tissue
e. Mouse mammary cancer cells
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Polymerase Chain Reaction
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• Polymerase Chain Reaction (PCR)– 1983, Kary Mullis
• Copies (“amplifies”)DNA in a test tube using the same type of chemistry that cells use to copy DNA– Exponential amplification of specific, short (usually
2,000 bp or less) sequences of DNA– Products are called amplicons
• Highly sensitive• Can amplify small quantities• Rapid and robust
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• Reaction ingredients:– PCR Primers
• Short, single-stranded DNA polynucleotides that are complementary to the sequences which flank the target region
• Made on DNA synthesizer
– dNTPs (in abundance)– Template DNA – DNA Polymerase
• Thermostable (e.g. Taq polymerase)
– MgCl2 and buffer
5’-acggacttactgattcagaca-3’
3’-ggcatcgattagctcgcatatcg-5’
dATP
dCTPdTTP
dGTP
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• PCR steps:– Denaturation (94 deg C)– Annealing (typically 50-60 deg C)
• Set just below melting temperature of primers• 4 + 2 rule
– Extension (72 deg C)• Optimum temp for taq polymerase
– Cycling (denaturation, annealing, extension)• Typically 20-30 times
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Figure 18.5
3′ 5′
(b) The 3 steps of a PCR cycle
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Many copiesof the gene ofinterest, flankedby the regionswhere theprimers bind.
Gene of interest
Chromosomal DNA
A different primerbinding near the otherend of the gene
Primer bindingnear one endof the gene
Many PCR cycles
(a) The outcome of a PCR experiment
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PCR is carried out in a thermocycler, which automates the timing of each cycle
All the ingredients are placed in one tube
The experimenter sets the machine to operate within a defined temperature range and number of cycles
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Figure 18.6
During each cycle, the DNAstrands are separated via heating.The temperature is then loweredto allow the primers to bind, anda complementary strand is made.
Region of interest that will be copied
Mix together template DNA,present in low amounts,with dNTPs, Taqpolymerase, and 2 primerspresent in high amounts.
Cycle 1
Cycle 2
Cycle 3
Template DNA
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Figure 18.6 The sequential process of
denaturing-annealing-synthesis is then repeated for many cycles
A typical PCR run is likely to involve 20 to 30 cycles of replication This takes a few hours to complete
After 20 cycles, a target DNA sequence will increase 220-fold (~ 1 million-fold) After 30 cycles, a target DNA sequence will increase 230-fold (~ 1 billion-fold)
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With each successivecycle, the relative amountof this type of DNA fragmentincreases. Therefore, aftermany cycles, the vastmajority of DNA fragmentscontain only the region thatis flanked by the 2 primers.
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Activity Cellular Replication PCR
Denaturation
Primers
Extension
Number of copies produced
Size of region copied
Ingredients
Purpose