central dogma of biology. dna replication dna structure

Central Dogma of Biology

DNA Replication

DNA Structure

DNA Replication

• is semi-conservative

• is bi-directional

• is semi-discontinuous

• initiation, elongation, termination

• contrasted in prokaryotes and eukaryotes

Is replication semi-conservative?

DNA Replication

• the two DNA parent strands can unzip (breaking weak H bonds)• the resulting single strands are “sticky” and can act as templates • template strands order the elongation of new strands• new strands are elongated by the addition of complementary dNTPs

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Three Hypotheses

Predictions After One Generation of Replication

Conservative Replication Refuted

Predictions After Two Generations

Dispersive Replication Refuted

Another test of semi-conservative replication

chromatid 1on left

chromatid 2on left

dNTP*

dNTP

dNTP

A

A B C D

chromatid 1on left

chromatid 2on right

dNTP*

dNTP

dNTP

A

chromatid 1on left

chromatid 2on left

dNTP*

dNTP

dNTP

A

Is replication unidirectional or bidirectional?

2 unzipping forks1 unzipping fork

Observation

Two PossibleInterpretations

unidirectional replication

bidirectional replication

Test: pulse-chase experiment

Allow replication over a short time course, with replacement of precursors:

cold,then hot (very radioactive), then warm (less radioactive)

unidirectional replication

bidirectional replication

predictions

observations

Is replication continuous?

Continuous Replication

The Problem

1. strands are antiparallel

5’

3’

3’

5’

nucleotide

Continuous replication requires that one strand be elongated in a 3’ to 5’ direction

3’

5’

5’3’

5’

3’

5’ 3’

2. but new strands are only synthesized in a 5’ to 3’ orientation

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3' to 5' strand elongation

5' to 3' strand elongation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

Why are lagging strands formed by fragments?

Alternatives: whole molecule unzips first before lagging strand is formed

Alternatives: lagging strand formed of small fragments that get connected

Fragment Formation

3' to 5' strand elongation

5' to 3' strand elongation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

Practice

Practice

5' to 3' strand elongation

3' to 5' strand elongation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

L S & L S & L

With Functional Ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

ContinuousSemi-Discontinuous

Discontinuous

With functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

observed

ContinuousSemi-Discontinuous

Discontinuous

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

5' to 3' strand elongation

3' to 5' strand elongation

With non-functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

ContinuousSemi-Discontinuous Discontinuous

With non-functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

observed

ContinuousSemi-Discontinuous Discontinuous

5' to 3' strand elongation

3' to 5' strand elongation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

Why are lagging strands formed by fragments?

Alternatives: whole molecule unzips first before lagging strand is formed

Alternatives: lagging strand formed of small fragments that get connected

Fragment Formation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

5' to 3' strand elongation

3' to 5' strand elongation

Practice

Practice

5' to 3' strand elongation

3' to 5' strand elongation

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

L S & L S & L

With Functional Ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

ContinuousSemi-Discontinuous

Discontinuous

With functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

observed

ContinuousSemi-Discontinuous

Discontinuous

Replication Hypotheses

Ligase+

Ligase-

Continuous Semi-Discontinuous Discontinuous

5' to 3' strand elongation

3' to 5' strand elongation

With non-functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

ContinuousSemi-Discontinuous Discontinuous

With non-functional ligase

amountof new

sequences (radioactivity)

W X

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Y

Sequence Size

Centrifuge Tube

top bottom

amountof new

sequences (radioactivity)

Sequence Size

Centrifuge Tube

top bottom

Z

observed

ContinuousSemi-Discontinuous Discontinuous

Initiation

Inititation at Origin of Replication

Primase and primer formation

Elongation

Elongation - 4 stepsRNA primer

elongation with dNTPs

previous fragment

ligation

elongation with dNTPswith upstream primer removal

Fragment Formation

DNA and RNA Polymerasesproofreading requires a previous nucleotide

A

G

C

T

A

A

A

T

T

A

G

C

T

A

A

A

U

U

DNAPolymerase

RNApolymerase elongation

proof-reading

elongation

DNA Proof-reading

A

G

C

T

A

A

G

T

T

A

G

C

T

A

A

T

TG

A

A

G

C

T

A

A

A

T

T

G

A

G

C

T

A

A

G

A

T

T

Proof-reading requires the presence of a previous nucleotide before a new one can be added …RNA polymerase has no such requirement (primase either)

Why are there RNA primers?

• New strands must be started in isolation (no 3’ OH on a previous nucleotide to act as an anchor)

• Only RNA polymerases (like primase) can begin in the open like that

• Why have no similar DNA polymerases evolved? Without proof-reading, a DNA primer would be a mutational hot-spot …better to have an RNA primer because the hot spot is more readily recognized (ribose nucleotides) and removed.

Replication Fork with Primosomesand Separate DNA Polymerases

Problems

• Kinetics of replication don’t reflect the kind of delay expected for disassociation and reassociation of the lagging polymerases

• SEM images show variable sized small loops associated with replication forks

Replication Fork with Replisomes

Replisome

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Why did DNA replace RNA as the hereditary molecule?

• Mutations usually include only one strand at first

• A double stranded nucleic acid stores information in the unaffected strand that can be used to correct mutations in the affected strand

DNA Repair

The double strandedness of DNA enables:

• recognition of mutation sites

• replacement of excised nucleotides with complementary nucleotides

Single Nucleotides or Whole Sequences Can Be Excised and Replaced