DNA is the Genetic Material Therefore it must 1.Replicate faithfully 2.Have the coding capacity to...

DNA is the Genetic Material

Therefore it must1. Replicate faithfully2. Have the coding capacity to

generate proteins and other products for all cellular functioning

• “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 replication1) 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)

1958: Matthew Meselson & Frank Stahl’s ExperimentSemiconservative model of DNA replication

DNA replication

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)

Isolated:Proteins from E. coli

+Template DNA

Added:- dNTPs (nucleotides) all 4 at once- Mg2+ (cofactor)- ATP (energy source)- free 3’OH end (primer)

Synthesized new DNA and isolated a DNA polymerizing enzyme

DNA polymerase I

3’

Kornberg was also able to characterizeHow the DNA polymerase worked!

- 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 strandsRun 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-discontinuousDNA Replication is Semi-discontinuous

Continuous Synthesis

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