1

Replication of the Genetic Material

Genetic material must be duplicated for transfer into

daughter cells.

Complementary double-stranded DNA

makes this possible

A

A

A

2

Possible Mechanisms for DNA Replication

Which is correct?

semiconservative conservative dispersive

3

Meselson - Stahl Experiment (1958)

Nitrogen isotopes

Density gradient centrifugation - CsCl

4

Meselson - Stahl Experiment

Definitely not conservative

Results also consistent with dispersive

5

Meselson - Stahl Experiment

Supports only semiconservative model

6

Replication of DNA in Eukaryotic Chromosomes

How is DNA arranged and replicated?

7

Replication of DNA in Eukaryotic Chromosomes

Taylor, Woods, and Hughes (1958)

CHO harlequinchromosomes

8

DNA Polymerase Chain Elongation

Chain growth - 5’ to 3’ direction

3’ 5’ 5’ 3’

9

DNA Polymerase Chain Elongation

10

Replication Origin

Bidirectional Replication from Origin

Single origin in

Prokaryotes

11

Replication Origins - Replicons

Multiple origins in Eukaryotes

Replicons

12

Primer Requirement

13

Initiation of DNA Replication - E. coli

Initiator protein DnaA binds oriC

denaturation

DNA helicases unwind DNA

DNA primase makes primers

14

Replication Fork - Okazaki Fragments

Initial primers

Repriming

Discontinuous synthesis Lagging strand

Continuous synthesisLeading strand

5’

5’3’

3’

5’

3’

5’

3’

15

Activities of DNA Polymerases

16

Types of DNA Polymerases

E. Coli DNA polymerase I - single polypeptideDNA polymerase III - three polypeptide holoenzyme

Both capable of chain growth and 3’-5’ exonuclease activity Only pol I has 5’ - 3’ exonuclease activity

Eukaryotes

> 15 polymerases, 3 involved in replication of nuclear DNA

pol - with primase, creates RNA/DNA primerpol and pol - extend primers on leading/lagging strand

17

Joining of Okazaki Fragments

18

DNA Replication in E. coli (Fig. 3.6)

Initiation

Elongation

Primer Removal

Ligation

19

DNA Replication in E. coli

Figure 3.8. Replisome Model. DNA pol III on lagging-strand templateis finishing synthesis of an Okazaki fragment.

20

Telomere Problem

Due to:Linear chromosomes & Requirement for RNA primers

21

Resolving the Telomere Problem

22

Rolling Circle Replication

23

Rolling Circle Replication in Phage