1. BIOL 2416 CH 3: DNA Replication

2. Fig. 3.1 Three models for the replication of DNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

3. Meselson-Stahl Experiment
Ruled out dispersive and conservative models of DNA replication in E. coli
Supported semi-conservative model
Used a density label/CsCl gradient and E. coli
See iGenetics animation
Semi-conservative replication confirmed in eukaryotes (CHO) with base analog 5-bromodeoxyuridine; causes lighter Giemsa
staining

5. DNA Replication
semi-conservative
bi-directional
semi-discontinuous

6. Fig. 3.4a DNA chain elongation catalyzed by DNA polymerase
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

7. III
III I I
DNA

8. DNA Polymerase: Needs:
Template
Primer (with free 3’ OH on the sugar)
All 4 dNTPs
Mg2+
Must work 5’ to 3’ only (extends at 3’OH end of growing DNA chain)
May have more than 1 function (domains)

9. Prokaryotic DNA Polymerases
TYPE
5’ to 3’
polymerase act’y
(forward writing)
3’ to 5’ exonuclease act’y
(backspace button)
exonuclease act’y
(forward erasing)
GENES I
yes
replace RNA primer w/ DNA
(and DNA repair)
proofread “fresh” DNA
remove RNA primers
(DNA repair)
polA II no
polB
III
extend primer w/ new DNA
10 genes (dnaE, dnaQ, holE core + 6 more polypeptides)

11. Prokaryotic DNA Replication
Initiator proteins bind to
Origin of Replication (oriC sequence)
DNA Helicase binds to initiator proteins, unwinds helix (causes +ve supercoils).
Single Stranded Binding Proteins keep DNA propped open.
Primase binds / forms RNA primer.

12. 5. DNA Pol III binds to 3’ RNA
primer; using its 5’ to 3’ polymerase (DNA writing) act’y extends new DNA (at 103bp/s);
proofreads with its 3’ to 5’ exonuclease act’y (backspace button).
6. DNA Gyrase (Topoisomerase II) relaxes +ve supercoils forming in front of fork.

13. DNA gyrase removes extra twists
Figure 7-19a

14. DNA gyrase removes extra twists
Figure 7-19b

15. Leading strand made in 1
piece (continuous)
Lagging strand
Moonwalks (discontinuous);
consists of
Many 1-2 kb Okazaki
fragments, each with own
~ 10 bp primer.
(Replicating DNA rotating
100 x / sec.)

16. DNA Pol I uses 5’ to 3’ exonuclease act’y to cut out RNA primers; uses its 5’ to 3’ Polymerase act’y to replace w/ DNA (also used to repair DNA). Uses 3’ to 5’ Exonuclease act’y to proofread as it goes along.
8. DNA Ligase “superglues” Okazaki fragments (backbone) together.

17. Fig. 3.10 Bidirectional replication of circular DNA molecules
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

18. Unusual DNA Replication
Rolling circle replication in plasmids and some Bacteriophage DNAs:
Nick
5’ end rolled out / bound by SSBs
Discontinuous replication (Okazaki fragments)
3’ end replaced on circle
Produces a linear daughter DNA
Telomeres in eukaryotes:
reverse transcriptase
roles in aging, cancer

19. Fig The replication process of double-stranded circular DNA molecules through the rolling circle mechanism
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

20. Fig. 3.18 The problem of replicating completely a linear chromosome in eukaryotes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

21. Fig. 3.19 Synthesis of telomeric DNA by telomerase
Telomerase elongates telomeres (new red bases):
Telomerase leaves; primase puts in new RNA primer;
DNA Polymerase elongates as usual:
Results in longer 5’ end thanks to telomerase
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

22. Eukaryotic DNA Replication
Must deal with histone/protein complexes: must temporarily disassemble nucleosomes, then reassemble
Slower: 50 bp/s
Many origins per chromosome; multiple replication bubbles that “pop” into each other (“replicons”)
During S phase of Interphase (growth phase), before mitosis or meiosis (cell division)
Different polymerases: 