Genetics Chapter 9

8/13/2019 Genetics Chapter 9

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DNA Replication

Chapter 9


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How is DNA synthesized?

Parental strand is used as a template for

the newly replicated strand


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3 possible models for DNA

replication

1. Semiconservative

2. Conservative

3. Dispersive


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Experimental evidence for

Semiconservative Model

Meselson and Stahl experiment (1958):

1. Incorporate heavy isotope of nitrogen(15N) into both strands of E. coliDNA,

then allow replication in medium

containing only the light isotope (14N)

2. Separate newly replicated DNA using

density centrifugation


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Predicted results for the 3 models

of DNA replication

(conservative

model

disproved)

(dispersive

model

disproved)


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DNA replication in eukaryotes is

also semiconservative

http://mol-biol4masters.masters.grkraj.org/html/Prokaryotic_DNA_Replication1-Introduction.htm

Herbert Taylors experiment (1958):


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Replicating Chromosome in

E. coli

The E. colichromosome is circular

Preserves the integrity of the circularchromosome

DNA replication initiates at a single

point and proceeds from one or two

replication forks

John Cairns (1963):


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DNA Replication in E. colistarts at an

origin and is bidirectional


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DNA replication in eukaryotes starts at

multiple origins and is bidirectional

Replication bubbles form

at mult ip leor ig inson thelinear eukaryotic

chromosome

replicon: DNA replicated

from a single origin

Drosophi lachromosomes


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DNA Synthesis

Requires DNA Polymerase

1. 3 DNA polymerases : I, II, and III

- polymerase III : replicates most of the DNA

2. Synthesizes DNA in a 5 to 3 direction

3. Synthesizes DNA antiparallel to the parental

strand


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Nucleotide Addition : 5 to 3 Synthesis

Catalyzes formation of

phosphodiester bond

between 5 PO4of

deoxyribonucleotide

and 3 OH on

DNA strand

- substrate is

deoxynucleotide triphosphate

Energy for polymerization comes from cleaving 2

phosphates from deoxyribonucleotide triphosphate

http://en.wikibooks.org/wiki/Medical_Physiology/Cellular_Physiology/DNA_and_Reproduction

5

5

3

5

3


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Nucleotide Addition : 5 to 3 Synthesis

Leading strand is synthesized

continuouslyin direction of

the movement of the

replication forkDNA pol III : high processivity

(synthesizes very long strand)

Lagging strand issynthesizeddiscontinuously(in pieces:

Okazakifragments) in

direction opposite of

movement of replication fork

leading strand

lagging strand


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Leading and Lagging Strand

Synthesis requires primers

Primersare short sequences

of nucleotides (RNA or DNA)

which are base paired to the

template DNA

Primers provide a de novo3OH end for an incoming

deoxyribonucleotide


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Leading and Lagging Strand

Synthesis at the Replication Fork

(~150bp in eukaryotes)


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Lagging Strand Synthesis

Four steps:

1. Primer synthesis

2. Elongation

3. Primer removal with gap filling

4. Ligation


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Primer Synthesis

Primasesynthesizes a primer (10-12 nucleotideslong) complementary to DNA template strand


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Elongation

DNA polymerase III adds deoxyribonucleotides to the3 OH end of the primer

E. coli :

400 nt per second !


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Proofreading activity of DNA polymerase3 to 5 exonuclease activity removes mismatched base pairs

- nuclease: degrades DNA- exonucleasecleaves from end

- endonucleasecleaves phosphodiester

bond between nucleotides

* every polymerase has 35 exonuclease

activity

** only DNA pol I has 53 exonuclease

activity


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Elongation

Fidelity: a measure of polymerase accuracy at incorporating

correct deoxynucleotide(E. coli : 1 mismatch in 109base pairs !)


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Primer Removal and Gap Filling

53 exonucleaseactivity of

DNA polymerase Iremoves primer

53 polymeraseactivity of

DNA polymerase I

fills in the gap with DNA

why is primer made of RNA ?

- primers are not always exact

matches to template

RNA primer can be removedand correct DNA sequence

added by pol III


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Ligation

DNA Ligase seals up nicks left after DNA

polymerase has filled in the gaps

ligase: catalyzes

formation of aphosphodoester

bond from a

5 phosphate

and 3 OH


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DNA synthesis begins at an origin

Step 1: Initiator proteins (dna A) bind origin (ori C)ori C : specific DNA sequence, ~245 bp long


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Binding of initiator proteins at

origin denatures DNA


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HelicaseUnwinds DNA, Primase

Synthesizes a Primer

Helicase + Primase = Primosome


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DNA Polymerase Synthesizes

the Leading Strand


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Primosomes generate more primers

for lagging strand synthesis

C ti d di ti DNA


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Continuous and discontinuous DNA

synthesis occur at replication fork

continuoussynthesisdiscontinuoussynthesis

At li ti f k i


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At replication fork : primosome +

2 DNA pol IIIs + ssb proteins

ssbsbind to single-stranded DNA and keep it from

reannealing during replication

holoenzyme: protein complex with

all associated subunits

DNA pol III : 10 subunits total

- 3 form core enzyme required

for activity

- subunit : clamp that holdspolymerase onto DNA

processivity

replisome:

primosome +

2 DNA pol IIIs

- move as a unit

along DNA


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polymerase cycling :

moving off and on the

lagging strand template

as Okazaki fragments

are completed

for overall movement in

direction of replication

fork :lagging strand must

loop through DNA pol III

to allow 53 synthesis

of Okazaki fragment

away from replication fork

E t t th li ti f k


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Events at the replication fork

during polymerase recycling

2. release of primase3. recruit clamp

1. synthesis of RNA primer

4. recruit DNA pol III,

Okazaki fragment

synthesis 53

5. reattach primase

further along

lagging strand


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Supercoiling

Topoisomerasescan put in or remove supercoils from the DNA

linkage number (L) : number of turns of 1 helix around the other

positive: circular DNA winds around

itself in the same direction asthe twist of helix (right handed)

negative: circular DNA winds around

itself in the opposite direction asthe twist of helix (left handed)


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Topoisomerases

Change the supercoiling of the DNAby increasing or decreasing the

linkage number

Type I Topoisomerases cut one of

the DNA strands, insert unbroken

end in opening

Type II Topoisomerases cut bothDNA strands, insert unbroken

double helix through opening

i.e.,gyrase


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Topoisomerases

Requiredfor DNA replication

As helix opens during DNA replication, positive

supercoils occur in front of the replication forkgyrase removes those supercoils so that

DNA replication can proceed


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Topoisomerase

Termination of
http://www.youtube.com/watch?v=EYGrElVyHnU&feature=relatedhttp://www.youtube.com/watch?v=EYGrElVyHnU&feature=related


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Termination of

Replication in E. coli

tersites (directly opposite oriC site)

bound by termination protein

(encoded by tusgene : termination

utilization substance)

Intertwined chromosomes have to

be separated by topoisomerase

(type II)

Oth d l t li t


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Other models to replicate

circular chromosomes

1. Rolling Circle

2. D loop


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Rolling Circle Replication in

E. coliPlasmids


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D-LoopReplication in

Mitochondria and Chloroplasts

origins are at different places

on parental template strand

unidirectional leading-strand

synthesis from both strands

(Displacement loop)

R t f DNA S th i d i


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Rates of DNA Synthesis during

replication

E.coli: 25,000 bp/min

eukaryotic : 2000 bp/min


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Eukaryotic DNA Replication

differences from prokaryotes :

9 different DNA polymerases

- : major one in replication

- : makes Okazaki fragment primers

RNA primers removed by RNAse, not DNA pol Multiple origins

- yeast : ARS (autonomously replicating sequences)

Telomeres: special sequences at chromosome ends

similarities to prokaryotes :

specific sequences at origins are bound by proteins

- called Origin Replication Complex (ORC) in eukaryotes

all of the enzymatic processes

How Are the Ends of


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How Are the Ends of

Chromosomes Preserved during

DNA Replication?


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Telomerase

1. Telomerase RNA base pairs with single-stranded 3 end

of DNA

2. Telomerase extends telomere by reverse transcription

(RNA from telomerase is template)

adds GGGGTT (or similar) sequence

3. Telomerase translocates to extended 3 end

RNA + enzyme complex that binds and extends 3 end of linear

chromosomes**ultimate goal : not to increase length, but preserve telomere

several

times

- then primase, polymerase, and ligase make DNA strand

complementary to the new telomere sequence


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Telomerase

1. Telomerase RNA base pairs with DNA


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Telomerase

2. Telomere extension occurs

reverse transcription


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Telomerase

3. Telomerase translocates to extended 3end


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Telomerase

4. Telomerase extends 3 end of telomerereverse transcription

How Telomerase Extends the 3 End


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How Telomerase Extends the 3 End

of a Linear Chromosome

exact size of telomere may

fluctuate from one round

of DNA replication to

the next, but . . .

** genomic information

adjacent to telomere

is preserved**

can be


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Telomere binding proteins

regulate telomeres

TRF1: # of proteins bound to telomeres determines whether

telomerase should extend telomeres

TRF2: prevent end-end fusion of different chromosomes

van Steensel et al., Cell 92::Pages 401413 (1998)


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DNA packaging and replication model
http://www.youtube.com/watch?v=OjPcT1uUZiE&feature=player_embeddedhttp://www.youtube.com/watch?v=OjPcT1uUZiE&feature=player_embedded

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Genetics Chapter 9