1. Lecture 24: DNA replicationG
0.34 nm
3.4 nm
1 nm
Figure 16.7a, c
(c) Space-filling model

2. Lecture Outline Overview of DNA replication
DNA has polarity (5’P, 3’OH)
Rules of Replication
Details of the replication machine
Proofreading
Mutation
Special problems at the ends of chromosomes C T A G
0.34 nm
3.4 nm
1 nm

3. Review Watson-Crick model of DNA structure Base composition ratiosG
0.34 nm
3.4 nm
1 nm
Watson-Crick model of DNA structure
Base composition ratios
A=T; G=C
Radioactive labeling experiments show that
DNA is the genetic material
Replication is semi-conservative

4. Chromosomes
Bacterial chromosomes tend to be circular, eukaryotic chromosomes linear
DNA plus associated proteins = chromatin
Euchromatin vs heterochromatin

6. DNA Structure N H O CH3 Sugar Adenine (A) Thymine (T) Guanine (G)
0.34 nm
3.4 nm
1 nm N H O
CH3
Sugar
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
Figure 16.8
5’ Carbon has PO4
3’ Carbon has OH

8. In DNA replication
The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
(a) The parent molecule has two complementary strands of DNA Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
(b) The first step in replication is separation of the two DNA strands.
(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T G
Figure 16.9 a–d

9. Replication overview
Must maintain integrity of the DNA sequence through successive rounds of replication
(think of the game “telephone”)
Need to:
unwind DNA, add an RNA primer, find an appropriate base, add it to the growing DNA fragment, proofread, remove the initial primer, fill in the gap with DNA, ligate fragments together
All of this is fast, about 100 bp/second
animation from DNAi

10. Elongating a New DNA Strand
DNA polymerases, add nucleotides to the 3 end of a growing strand
New strand
Template strand
5 end
3 end
Sugar A T
Phosphate
Base C G G C A T P P P C OH
Figure 16.13
5 end
Nucleoside
triphosphate P P
Pyrophosphate

11. Template 3’ 5’ 3’ 5’ Primer with a free 3’ OH P C OH T G A P G P T P A

12. Rules of Replication Semi-conservative Primer is required
Template is required
Elongation occurs 5’ -> 3’ 
Semi-discontinuous
Bidirectional

13. DNA synthesis Needs a template Must add a base to an existing 3’ OH
This end has a 5’ phosphate
Needs a template
Must add a base to an existing 3’ OH
This end has a 3’ OH
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

14. Circular bacterial chromosomes have one origin
Eukaryotic chromosomes have hundreds or even thousands of replication origins
Close-up of a replication fork
Origin of replication
Parental (template) strand
0.25 µm
Daughter (new) strand
Bubble
Replication fork
Two daughter DNA molecules

15. Overall direction of replication
Synthesis of leading and lagging strands during DNA replication
Parental DNA
DNA pol Ill elongates
DNA strands only in the
5  direction. 1
Okazaki
fragments
DNA pol III
Template
strand
Lagging strand 3 2
DNA ligase
Overall direction of replication
One new strand, the leading strand,
can elongate continuously 5 
as the replication fork progresses.
The other new strand, the
lagging strand must grow in an overall
3  direction by addition of short
segments, Okazaki fragments, that grow
5  (numbered here in the order
they were made).
DNA ligase joins Okazaki
fragments by forming a bond between
their free ends. This results in a
continuous strand. 4
Figure 16.14 3 5
Leading strand

16. Model for the “replication machine,” or replisome

17. Model for the “replication machine,” or replisome
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

18. Detail of the lagging strand
Primase joins RNA nucleotides into a primer. 1
Overall direction of replication 3 5 1 2
Template strand
RNA primer
Okazaki fragment
Figure 16.15
DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 2
After reaching the next RNA primer (not shown), DNA pol III falls off. 3
After the second fragment is primed. DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 4
DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5
DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 6
The lagging strand in this region is now complete. 7

19. First look at leading strand:
Helicase unwinds a bit of DNA
Primase adds an RNA primer
Polymerase adds complementary nucleotides to 3’ OH and slides down the molecule, continuing to extend the sugar-phosphate chain.
(E. coli has about 40,000 turns in the circular chromosome-- each of them must be unwound to separate the DNA strands)

20. Now look at lagging strand
Can’t form continuous molecule, since synthesis only happens 5’ to 3’. SO, need many primers and lots of short fragments
Primase adds an RNA primer and Polymerase adds nucleotides until it reaches the previous primer
Another polymerase removes RNA primer on adjacent stand and adds dNTPs
Ligase connects the fragments

21. Table 16.1 Bacterial DNA replication proteins and their functions

22. Try this at home:
Draw a replication bubble, label the bases on the template, primer, and newly synthesized DNA using the shorthand DNA notation.
Convince yourself that synthesis must go 5’ to 3’ and that replication must be semi-discontinuous.