Broad Course Objectives for DNA Replication Students will be able to: describe the historic experiment that demonstrated DNA replication follows a semi-conservative

Broad Course Objectives for DNA Replication

Students will be able to:• describe the historic experiment that demonstrated

DNA replication follows a semi-conservative model.• describe the process of DNA replication in

prokaryotes at the biochemical level• explain how proofreading and repair is accomplished

during DNA synthesis

Outline/study guide—DNA Replication

• At what point in the cell cycle does DNA replication occur?

• When two DNA molecules (or chromosomes) are made from one, where do the parental strands end up, vs. the newly synthesized strands? (i.e. semiconservative replication)

• Why can DNA only be synthesized in the 5’ 3’ direction?

• What are the enzymes and proteins involved in DNA synthesis? What is the function of each and at what point do they act?

• At what point does RNA function in DNA replication?• What determines the lagging strand vs. the leading strand? How does

this change on the “other” side of the replication origin?• How are the Okazaki fragments joined into one continuous DNA strand?

• How does the DNA replication machinery correct errors made during replication?

• Are human chromosomes linear or circular? Bacteria?• Why do linear chromosomes (but not circular chromosomes) have a

problem with telomeres becoming shorter and shorter with each round of replication? How do some cells get around this?

48 year old woman with Werner Syndrome

Progeria

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

CA

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

3′ 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

Brooker Fig 13.1

Identical base sequences

Each strand of the parent DNA

molecule becomes a

template for the new

molecule(s)

The width of the nucleotides reflect larger purines and smaller pyrimidines

Brooker fig 13.2

Conservative model

Firstreplication

Second replication

OriginalDNA

Semiconservative model Dispersive model


Different models of DNA Replication

Brooker fig 13.2

Conservative model

FirstReplication (N14)

Second Replication(N14)

OriginalDNA (N15)

Semiconservative model Dispersive model


How do you expect the different models to appear in the centrifuge experiment?

Brooker fig 13.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Experimental level Conceptual level

2. Incubate the cells for various cell generations

3. Lyse cells to release DNA

4. Load sample of lysate onto CsCl gradient.

5. Centrifuge until the DNA molecules reach equilibrium densities.

6. View DNA within the gradient using a UV light.

DNACell wall

Cell membrane

Light DNA

Half-heavy DNA

Heavy DNA

(after 2 generations.)

CsClgradient

Lysate

37°C

14Nsolution

Suspension ofbacterial

cells labeledwith 15N

Up to 4 generations

Density centrifugation

Generation0

1

Add 14N

2

1. Grow bacteria in excess of 15N-containing compounds. Switch to 14N at Generation 1.

15N-DNA = purple 14N-DNA = blue

Experiment to distinguish between DNA replication models

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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

After one generation, DNA is “half-heavy”(consistent with both semi-conservative and dispersive models)

After ~ two generations: DNA is “light” and “half-heavy”(Consistent with which model?)

Why does DNA (and RNA) only “grow” in the 3’ direction?

New strand Template strand

5’ end 3’ end

Sugar A TBase

C

G

G

C

A

C

T

PP

P

OH

P P

5’ end 3’ end

5’ end 5’ end

A T

C

G

G

C

A

C

T

3’ end

Nucleosidetriphosphate

Pyrophosphate

2 P

OH

Phosphate

Fig from Cambell and Reece, 7th ed(Like Brooker, fig 13-15)

Brooker, fig 13.10

Origin of replication

Replicationforks

Direction ofreplication fork

1st Okazakifragment

First and second Okazakifragments have beenconnected to each other.

1st Okazaki fragmentof the lagging strand

2nd Okazakifragment

3rdOkazakifragment

Primer

Primer

The leading strand elongates,and a second Okazaki fragmentis made.

The leading strand continues to elongate. A third Okazaki fragment is made, and the firstand second are connected together.

Primers initiate DNA synthesis.Synthesis of the leading strand occurs inthe same direction as movement of thereplication fork. 1st Okazakifragment of lagging strand ismade in 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

Overview of DNA

Replication


Brooker, fig 13.7

5′3′

5′

5′

3′

3′DNA polymerase III

Origin

Leadingstrand

Lagging strand

Linked OkazakifragmentsDirection of fork movement

DNA polymerase III

RNAprimer

Okazaki fragment

DNAligase

RNA primer

Single-strandbinding protein

DNA helicase

Topoisomerase II

Parental DNA

Primase

Replication fork

• DNA helicase breaks the hydrogen bonds between the DNA strands.

• Topoisomerase alleviates positive supercoiling.

• Single-strand binding proteins keepthe parental strands apart.

• Primase synthesizes an RNAprimer.

• DNA polymerase III synthesizes adaughter strand of DNA.

• DNA polymerase I excises theRNA primers and fills in withDNA (not shown).

• DNA ligase covalently links theOkazaki fragments together.

Functions of key proteins involved with DNA replication

Brooker, fig 13.5

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′

Origin of Replication

Brooker fig 13.6

AT-rich region DnaA boxes

DnaA proteins bind to DnaA boxes and toeach other. Additional proteins that causethe DNA to bend also bind (not shown).This causes the region to wrap aroundthe DnaA proteins and separates theAT-rich region.

DNA helicase

DNA helicase (DnaB protein) binds to theorigin. DnaC protein (not shown) assiststhis process.

DNA helicase separates the DNA in bothdirections, creating 2 replication forks.

DnaA protein


5′ 3′

AT- rich

region

ForkFork

3′ 5′

5′3′

5′3′

5′3′

3′5′

3′5′

3′

5′

How the origin sequence initiates

replication

DNA helicase

DNA helicase separates the DNA in bothdirections, creating 2 replication forks.

ForkFork

5′3′

5′3′

3′5′

3′

5′

Brooker fig 13.6

Travels along the DNA in the 5’ to 3’ direction

Bidirectional replication


Replication initiation cont.


Brooker, fig 13.8

3′

3′ exonucleasesite3′

5′ 5′

Fingers

Thumb

DNA polymerasecatalytic site

Templatestrand

Palm

IncomingDNA nucleotides (triphosphates)(dNTPs)

Schematic side view of DNA polymerase III (bacterial)

Model for how the leading strand and lagging strand coordinate at the replication fork

Brooker Fig 13.12

5′

3′5′

3′

3′

5′ 5′

DNA helicase

Replisome

Primosome

Topoisomerase

Leading strand

DNApolymerase III

Single-strandbinding proteins

Regionwherenext Okazakifragmentwill be made

Primase

RNA primer

New Okazakifragment

Older Okazakifragment

Replicationfork

5′


Orientation of lagging strand in the replication bubble

Brooker, ch 13

Chromosome Sister chromatids

Before S phase During S phase End of S phase

Origin

Origin

Origin

Origin

Origin

Centromere(DNA under thekinetochore)


Eukaryotes have hundreds of origins of replication on their (linear) chromosome

Replicating DNA of Eukaryotic Chromosomes (Drosophila melanogaster)

Fig from iGenetics, Russell

Brooker, fig 13.4a

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

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ht

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raw

-Hil

l Com

pan

ies,

In

c. P

erm

issi

on r

equ

ired

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From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press.

Replicationfork

Bacteria only have one origin on their (circular) chromosome

Replication rate

• Eukaryotic DNA replication– Typical human chromosome length: 100 million bp– Time to replicate a chromosome: minutes to hours– Hundreds of origins per chromosome– Replicon = ~20,000 to 300,000 bp long– 500-5000 bp / minute at each replication fork

(slower than bacterial replication; that much harder to “unwind” the DNA for replication).

• Bacterial (prokaryotic) replication: – Single circular chromosome (~4.6 million base pairs

[bp])– Single origin of replication single replicon

(“Replication Bubble”)

Requirements of DNA Replication in a complex organism

• Very low error rate:– One human cell: 6 billion bp of DNA. A

copying error rate of 1 error/million nt 6000 errors with every cell division

• Very fast copy rate– E. coli –1000 nt per minute 3 days to

replicate (real life: 20 minutes per cell cycle; 1000 nt per second)

Brooker, fig 13.21

DNA polymerase cannot linkthese two nucleotides togetherwithout a primer.

No place for a primer

3′

5′

Linear chromosomes (eukaryotic) cannot easily replicate the ends of chromosomes


Brooker, fig 13.20

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′

Chromosome gets shorter at the telomeres with each replication if overhang is left.

In humans and most complex organisms, telomerase is only used in continuously dividing stem cells (e.g. spermatogonia stem cells) most cells get shorter telomeres over time (age). What happened to Dolly, the cloned sheep? (she was generated from a skin cell with shorter telomeres, and she aged early)

Linear chromosomes (eukaryotic) must fill in gap left by RNA primer

Telomere

Telomerase

Eukaryoticchromosome

Repeat unit

3′

3

5′

T T A G G G T T A

A A T C C C A A TC C C A A U C C C

G G G A G G GT T A T TG G G

T T A G G G T T A

CC C A A U C C C

G G G T T A T T G

GG

T T AG G G A G G G

C C C A A U C C C

TT

C C C A A T A A A AT C C C U A AC UC CC C C

T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G

T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G GT T A G G

A A T C C C A A T

A A T C C C A A T

A A T C C C A A T

RNA

RNA primer

Telomerase synthesizesa 6-nucleotide repeat.

Telomerase moves 6nucleotides to the right andbegins to make another repeat.

The complementarystrand is made by primase,DNA polymerase, and ligase.

3′ 5′5′ 3′

3′


Brooker, figure 13.22

Step 1 Binding

Step 3 Translocation

The binding-polymerization-

translocation cycle occurs many times

This greatly lengthens one of the strands

Step 2 Polymerization

The end is now lengthened

How telomerase “finishes” the replication of

linear chromosomes

Go over lecture outline at end of lecture

Concept Checks

• In the Meselson and Stahl experiment, how was switching the bacterial media from N15 to N14 important for supporting the Semi-conservative model?

Concept check

• What are the functions of the A-T rich region and DNA boxes in the Origin of Replication?

Concept Check

• Why is primase needed for DNA replication?

• Is the template strand read in the 5’ to 3’ direction or the 3’ to 5’ direction?

Concept Check

• Describe the differences between Dna synthesis in the leading strand vs. the lagging strand.

Which component functions immediately after ligase?

a.Helicase

b.DNA Polymerase 1

c.DNA Polymerase 3

d.primase

e.none of the above

Which component functions immediately after ligase?

a.Helicase

b.DNA Polymerase 1

c.DNA Polymerase 3

d.primase

e.none of the above

Documents

Broad Course Objectives for DNA Replication Students will be able to: describe the historic experiment that demonstrated DNA replication follows a semi-conservative