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