lecture 2 (chapter 11) dna replication 1. “it has not escaped our notice that the specific pairing...

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

Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.

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

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


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


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′


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


40


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


Termination of Replication

45

Fork

Fork

Fork

Fork

ter(T2)

oriC

oriC

(T1)

Tus

Tus


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


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


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′


Base pair mismatch near the 3′ end

3′

3′

Incorrectnucleotideremoved

exonucleasesite

51

52


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


58

Figure 11.19


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

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


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′


68

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

70

We’ll demonstrate this on the board

71

End replication problem

http://www.google.com/url?sa=i&rct=j&q=end+replication+problem&source=images&cd=&cad=rja&docid=Cu4C4wcZsOm-_M&tbnid=d9YPOi7RGbNOjM:&ved=0CAUQjRw&url=http%3A%2F%2Fahcliposomes.com%2Fuploads%2FTA-65.pptx&ei=AyQgUb3bN6iDjAKp-YGAAQ&bvm=bv.42661473,d.cGE&psig=AFQjCNHJaX6-i8AEkSBGYnK3IA0GdvhsTg&ust=1361147245050505

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

72

74

Death phase is due to running out of food

75

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

76

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!

78

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

79

Polymerase Chain Reaction

80

81

• 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

82

• 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

83

• 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

Figure 18.5

3′ 5′

(b) The 3 steps of a PCR cycle


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

85


86

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


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

+

+

+

+

87

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)


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.

+

+

+

+

+

+

+


88

89

Activity Cellular Replication PCR

Denaturation

Primers

Extension

Number of copies produced

Size of region copied

Ingredients

Purpose

lecture 2 (chapter 11) dna replication 1. “it has not escaped our notice that the specific pairing...

Documents