Replication

RNA Synthesis

Decoding the Genetic Code

Noel Murphy

Reference Sources

Hartl & Jones, Genetics: Analysis of Genes and Genomes, 6th Edition

Chapter 6 – ReplicationChapter 10 – Transcription and the code

Klug & Cummings, Essentials of Genetics 5th Edition

Chapter 11 – ReplicationChapter 13 – Transcription and the code

Lectures http://www.tcd.ie/Genetics/staff/Noel_Murphy.htm

DNA is the Genetic Material

Therefore it must

(1) Replicate faithfully.

(2) Have the coding capacity to generate proteins and other products for all cellular functions.

• “A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.”

• -Francis Crick

Replication

The Dawn of Molecular Biology

April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."

Models for DNA replication

1) Semiconservative model:Daughter DNA molecules contain one parental strand and one newly-replicated strand

2) Conservative model:Parent strands transfer information to an intermediate (?), then the intermediate gets copied.The parent helix is conserved, the daughterhelix is completely new

3) Dispersive model:Parent helix is broken into fragments, dispersed, copied then assembled into two new helices.New and old DNA are completely dispersed

(a) Hypothesis 1:

Semi-conservative replication

(b) Hypothesis 2:Conservative replication

Intermediate molecule

(c) Hypothesis 3:Dispersive replication

MODELS OF DNA REPLICATION

Testing Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)

Meselson and Stahl Semi-conservative replication of DNA

Isotopes of nitrogen (non-radioactive) were used in this experiment

Generations

0

0.3

0.7

1.0

1.1

1.5

1.9

2.5

3.0

4.1

0 and 1.0 mixed

0 and 4.1 mixed

HH

HL

LL + HL

HH

HL

HL LL LL LH

Equilibrium Density Gradient Centrifugation

Detection of semiconservative replication in E. coli by density-gradient centrifugation. The position of a band of DNA depends on its content of 14N amd 15N. After 1.0 generation, all the DNA molecules are hybrids containing equal amounts of 14N and 15N

DNA replication Nucleotides are successively added using deoxynucleoside triphosphosphates (dNTP’s)

Replication as a process

• Double-stranded DNA unwinds.

The junction of the unwound

molecules is a replication fork.

A new strand is formed by pairing complementary bases with theold strand.

Two molecules are made.

Each has one new and one old

DNA strand.

DNA Replication

• Since DNA replication is semiconservative, therefore the helix must be unwound.

• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.

Origin

5’3’

3’5’

UNIDIRECTIONAL REPLICATION

Origin

5’3’

3’5’

BIDIRECTIONAL REPLICATION

Replication can be Uni- or Bidirectional

John Cairns

Grow cells for several generationsSmall amounts of 3H thymidineare incorporated into new DNA

Grow for brief period of time

Add a high concentration

of 3H- thymidine

in media with lowconcentration of

3H- thymidine

Bacterial culture

*T

*T

*T

*T

Dense label at the replication forkwhere new DNA is being made

*T*T *T *T

*T*T

*T*T

*T*T*T

*T*T

*T*T*T

*T*T *T *T

*T*T*T*T

*T*T*T

All DNA is lightlylabeled with radioactivity

*T*T *T

Cairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months.

Evidence points to bidirectional replication

Label at both replication forks

Features of DNA Replication

• DNA replication is semiconservative– Each strand of both replication forks is being

copied.

• DNA replication is bidirectional– Bidirectional replication involves two

replication forks, which move in opposite directions

Arthur Kornberg (1957)

Protein extracts from E. coli+

Template DNAIs new DNA synthesized??

- dNTPs (substrates) all 4 at once- Mg2+ (cofactor)- ATP (energy source)- free 3’OH end (primer)In vitro assay for DNA synthesis

Used the assay to purify a DNA polymerizing enzymeDNA polymerase I

3’

Kornberg also used the in vitro assay to characterizethe DNA polymerizing activity

- dNTPs are ONLY added to the 3’ end of newly replicating DNA

-therefore DNA synthesis occurs only in the5’ to 3’ direction

3’

3’

5’3’5’

5’3’5’

5’3’5’

5’3’5’ 3’

Parental template strandNew progeny strand

THIS LEADS TO A CONCEPTUAL PROBLEM

Consider one replication fork:

5’

3’

5’

3’

Direction ofunwinding

Continuous replication

5’

3’Primer

Primer

5’

3’

Primer

5’

3’Discontinuous replication

Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)

Evidence for Semi-Discontinuous Replication(pulse-chase experiment)

Bacteria arereplicating

Bacterial culture

Add 3H Thymidine

For a SHORT time(i.e. seconds)

Flood with non-radioactive T

Allow replicationTo continue

Harvest the bacteriaat different timesafter the chase

Isolate their DNASeparate the strands(using alkali conditions)Run on a sizing gradient

smallest

largest

Radioactivity will onlybe in the DNA that was made during the pulse

smallest

largest

Results of pulse-chase experiment

Pulse

5’

3’

5’

3’

Direction ofunwinding

3’

5’

Primer

Primer

5’

3’

Primer

5’

3’

* * *

***

Chase

Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous

Features of DNA Replication

• DNA replication is semiconservative– Each strand of template DNA is being copied.

• DNA replication is bidirectional– Bidirectional replication involves two replication forks,

which move in opposite directions

• DNA replication is semidiscontinuous– The leading strand copies continuously

– The lagging strand copies in segments (Okazaki fragments) which must be joined

The Enzymology

• In 1957, Arthur Kornberg demonstrated the existence of a DNA polymerase - DNA polymerase I

• DNA Polymerase I has THREE different enzymatic activities in a single polypeptide:

• a 5’ to 3’ DNA polymerizing activity

• a 3’ to 5’ exonuclease activity

• a 5’ to 3’ exonuclease activity

of DNA Replication

Subsequenthydrolysis ofPPi drives thereaction forward

Nucleotides are added at the 3'-end of the strand

The 5’ to 3’ DNA polymerizing activity

Why the exonuclease activities?

• The 3'-5' exonuclease activity serves a proofreading function

• It removes incorrectly matched bases, so that the polymerase can try again.

Proof reading activityof the 3’ to 5’ exonuclease.

DNAPI stalls if the incorrect ntd is added - it can’t add thenext ntd in the chain

Proof reading activity is slowcompared to polymerizingactivity, but the stalling ofDNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizingactivity and remove theincorrect base.

How?1) Base-pairing specificity at the active site- correct geometry in the active site occurs only with correctly paired bases BUT the wrong base still gets inserted 1/ 104 -105 dNTPs added2) Proofreading activity by 3’-5’ exonuclease- removes mispaired dNTPs from 3’ end of DNA- increases the accuracy of replication 102 -103 fold3) Mismatch repair system- corrects mismatches AFTER DNA replication

DNA Replication is Accurate(In E. coli: 1 error/109 -1010 dNTPs added)

Is DNA Polymerase I the principal replication enzyme??

In 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I

activity (polA)

- mutant was super sensitive to UV radiation

- but otherwise the mutant was fine i.e. it could divide, so obviously it can replicate its DNA

Conclusion:

• DNAP I is NOT the principal replication enzyme in E. coli

- DNAP I is too slow (600 dNTPs added/minute – would take 100 hrs to replicate genome instead of 40 minutes)

- DNAP I is only moderately processive(processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template)

Conclusion: • There must be additional DNA polymerases.• Biochemists purified them from the polA mutant

Other clues….

- functions in multiple processes that require only short lengths of DNA synthesis

- has a major role in DNA repair (Cairns- deLucia mutant was UV-sensitive)

- its role in DNA replication is to remove primers and fill in the gaps left behind

- for this it needs the nick-translation activity

So if it’s not the chief replication enzyme then what does DNAP I do?

A total of 5 different DNAPs have been reported in E. coli

• DNAP I: functions in repair and replication • DNAP II: functions in DNA repair (proven in

1999)

• DNAP III: principal DNA replication enzyme • DNAP IV: functions in DNA repair (discovered in

1999)

• DNAP V: functions in DNA repair (discovered in 1999)

The DNA Polymerase Family

The "real" replicative polymerase in E. coli

• It’s fast: up to 1,000 dNTPs added/sec/enzyme

• It’s highly processive: >500,000 dNTPs added before dissociating

• It’s accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall.

DNA Polymerase III