LE 16-7

LE 16-7

5 end

3 end

5 end

3 end

Space-filling modelPartial chemical structure

Hydrogen bond

Key features of DNA structure

0.34 nm

3.4 nm

1 nm

The mechanism of DNA Replication

When during the cell cycle is DNA synthesized? Draw

The Basic Principle: Base Pairing

• Each strand acts as a template for building a new strand in replication

• Parent dsDNA molecule unwinds & base pairs are broken- two new daughter strands built based on

base-pairing rulesDraw

LE 16-9_1

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

LE 16-9_4

The nucleotides are connected to form the sugar-phosphate back-bones of the new strands.

The first step is separation of the two parental DNA strands.

Synthesis of complementary strands

Predicted by Watson and CrickSemiconservative model ofDNA replication

A simple model of DNA replication

LE 16-10

Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.

Semiconservative model. The two strands of the parental moleculeseparate, and each functions as a template for synthesis of a new, comple-mentary strand.

Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

Parent cellFirstreplication

Secondreplication

Variousproposedmodels ofDNAreplication

• Meselson and Stahl experimentally supported

one of the replication models

How & which one?

LE 16-11

Bacteriacultured in mediumcontaining15N

DNA samplecentrifugedafter 20 min(after firstreplication)

DNA samplecentrifugedafter 40 min(after secondreplication)

Bacteriatransferred tomediumcontaining14N

Lessdense

Moredense

Conservativemodel

First replication

Semiconservativemodel

Second replication

Dispersivemodel

Heavy radioisotope

Light radioisotope

Why labelnitrogen?

Supported by data

• Replication begins– at origin of replication (ori)

• Creation of replication bubble with replication forks at each end (Draw)

•Hundreds to thousands of oris on eukaryotic chromosome• Usually one on bacterial chromosome

•Proceeds in both directions from each origin, until the entire molecule is copied

LE 16-12

In eukaryotes, DNA replication begins at many sitesalong the giant DNA molecule of each chromosome.

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand0.25 µm

Replication fork

Origin of replication

Bubble

In this micrograph, three replicationbubbles are visible along the DNAof a cultured Chinese hamster cell(TEM). Arrowheads mark replication forks.

Elongating a New DNA Strand

• Basic componentsTemplate DNA

DNA polymerase

DNA precursors deoxynucleotide triphosphates(dATP, dCTP, dGTP,dTTP)

LE 16-13

New strand5 end

Phosphate BaseSugar

Template strand3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate

5’

Specificity of DNA polymerase• only adds nucleotides to the free

3hydroxyl end of dsDNA

• New DNA strand made only in 5’-3’direction

Draw

LE 16-14

Parental DNA

5

3

Leading strand

35

3

5

Okazakifragments

Lagging strand

DNA pol III

Templatestrand

Leading strand Lagging strand

DNA ligase Templatestrand

Overall direction of replication

primer

LE 16-16

5

3Parental DNA

3

5


DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol ILaggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

LaggingstrandOrigin of replication

Other components of the DNA replication machinery?

DNA helicase- to unwind DNA

Single strand binding proteins- to stabilize ssDNA

DNA ligase- to seal gap in sugar-phosphate backbone (make phosphodiester bond) between Okazaki fragments

LE 16-15_1

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3


A Closer Look at Lagging Strand Synthesis

LE 16-15_2

53


Templatestrand

5 3


RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

LE 16-15_3

53


Templatestrand

5 3


RNA primer3

5

35


Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

LE 16-15_4

53


Templatestrand

5 3


RNA primer3

5

35


Okazakifragment

3

5

5

3



33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

LE 16-15_5

53


Templatestrand

5 3


RNA primer3

5

35


Okazakifragment

3

5

5

3



33

5

5


33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

LE 16-15_6

53


Templatestrand

5 3


RNA primer3

5

35


Okazakifragment

3

5

5

3



33

5

5


33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

33

5

5

DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.

The lagging strand in the regionis now complete.

Animation: Lagging Strand

Animation: DNA Replication Review

Proofreading and Repairing DNA

• DNA polymerases proofread• Replace mismatched nt in new DNA

• Also1. Mismatch repair: repair enzymes correct

errors in base pairing

2. Nucleotide excision repair: enzymes cut out and replace damaged stretches of DNA

Example

DNA exposure to ultraviolet (UV) lightinduces chemical crosslinks between adjacent thymines (thymine dimers)

How to repair?

LE 16-17

DNA ligase

DNA polymerase

DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.Nuclease

A thymine dimerdistorts the DNA molecule.

Is DNA replication of linear chromosomes ever complete?

Consider the tips (ends) of the leading and lagging strands.

LE 16-18

End of parentalDNA strands

5

3

Lagging strand 5

3

Last fragment

RNA primer

Leading strandLagging strand

Previous fragment

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase5

3

Removal of primers andreplacement with DNAwhere a 3 end is available

Second roundof replication

5

3

5

3Further roundsof replication

New leading strand

New leading strand

Shorter and shorterdaughter molecules

• Ends of eukaryotic chromosomes– Tipped with many copies of a short DNA repeat

called telomeres (e.g.human telomere sequence TTAGGG x 100-1,000)

• Added by telomerase , a ribozyme (made of RNA and proteins)

Function:Telomeres postpone loss of important genes near ends after each cell division.

Is telomerase found in all eukaryotic cells?

NO, mostly in germ cells but NOT in somatic cells.

What will happen to DNA in cells that continually dividesuch as epithelial cells (skin, gut)?

Make a prediction about the length of chromosomes in skin cellsfrom a 80 year old versus a 4 year old.

Cancer cells are characterized in part by their continuous cell division. Shouldn’t they ultimately die from loss of genes due to shortening of chromosomes?

Hypothesize why they continue to divide without injury?

Cancer cells express telomerase, which prevents

chromosome shortening

LE 16-19

1 µm

Labelled telomeres

Questions?

LE 16-7

Documents

Transcript of LE 16-7