THE EUKARYOTIC CELL CYCLE

S phase
DNA synthesis
G1
DNA replication Growth phase 1 Main checkpoints

and repair
G2
Go M
Growth phase 2
Quiescent Mitosis

1
 REPLICATION
START REPLICATION – GENERAL
Must be ready
PRINCIPLES
Must all start at the same time
Must know where to start ACCURACY/FIDELITY
Proof reading/repair
FINISH Must distinguish between original and copy

Must all finish

Must ensure that each piece of DNA is replicated
only once
Therefore must know where to finish

2
 REPLICATION
START REPLICATION – GENERAL
Must be ready : G1
PRINCIPLES
Must all start at the same time G1  S
Must know where to start ACCURACY/FIDELITY
ORIGIN OF REPLICATION
Proof reading/repair G2
FINISH Must distinguish between original and copy
EPIGENETICS
Must all finish complete S
Must ensure that each piece of DNA is replicated
only once
Therefore must know where to finish

3
 DNA polymerase DNA Polymerase - Matches the
correct nucleotides then joins
adjacent nucleotides to each other
Provides an RNA
primer to start
polymerization

Unwinds
the DNA
Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008)
and melts
it
Keep the
DNA single
stranded
DNA synthesis always occurs by adding after it has
nucleotides to the 3’-OH of the growing strand. been melted
by helicase
Synthesis is always in the 5’- 3’ direction

4
 Replication Enzymes
• DNA Polymerase - Matches the correct
nucleotides then joins adjacent
nucleotides to each other
• Primase - Provides an RNA primer to
start polymerization
• Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
• Helicase - Unwinds the DNA and melts it
• Single Strand Binding Proteins - Keep
the DNA single stranded after it has been
melted by helicase
• Gyrase - A topoisomerase that Relieves
torsional strain in the DNA molecule
• Telomerase - Finishes off the ends of
DNA strands
5
 Causes of DNA Damage
• Chemical mutagens
• Radiation
• Free radicals

Figure 5-41 Molecular Biology of the Cell (© Garland Science 2008)

6
 RADIATION = ENERGY RADIOLYSIS OF WATER

H2O . .
OH + H + e-

ENERGY DEPOSITION IN DNA S-A…….T-S

.
OH P
S-A…….T-S

S-A…….T-S
P
P
S-A…….T-S

A-S
P

P
P

P P
S-T…… A-S P C-S
P P S P
S-G……C-S P G-S
P

P
S-C….....G-S

S-T……..A-S
P

P
.OH
P

P
S-G

S-C

S-T
S
P

S-A……..T-S P
DNA DAMAGE P P
P
S-A……..T-S
P

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RESPIRATION AND AGEING

Carbohydrate + O2

Energy + CO2 + H2O

.02 H 2O 2 .
OH

DNA damage
~10000 lesions/cell/day
Maynard et al, 2008

8
 Types of DNA Damage: Base Loss and Base Modification Adenine tautomer

Chemical Modification Photodamage thymine dimer

Depurination

Deamination Chemical Modification by O2 free radicals

9
 Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
Generation of a • Correct nucleotide has greater affinity for
mutation by the moving polymerase than incorrect nucleotide
adenine • Exonucleolytic proofreading of DNA
polymerase
tautomer – DNA molecules with mismatched 3’ OH end are not effective
templates; polymerase cannot extend when 3’ OH is not base
- About every 1 paired
in 104 bases – DNA polymerase has separate catalytic site that removes
unpaired residues at terminus

10
 Maintenance of DNA Sequences
DNA Polymerase as Self Correcting Enzyme
Two catalytic sites

11
 DNA damage repair pathways.

DNA repair mechanisms in cells

• Base-excision repair (PARP)
• Nucleotide-excision repair
• Recombinational repair (BRCA1/BRCA2)
• Mismatch repair

Jalal S et al. Clin Cancer Res 2011;17:6973-6984

©2011 by American Association for Cancer Research

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 DNA damage repair pathways.
DNA Repair
Base Excision Repair
a. DNA glycosylase recognizes
damaged base
b. Removes base leaving deoxyribose
sugar
c. AP endonuclease cuts
phosphodiester backbone
d. DNA polymerase replaces missing
nucleotide
e. DNA ligase seals nick

Jalal S et al. Clin Cancer Res 2011;17:6973-6984

©2011 by American Association for Cancer Research

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Poly (ADP-ribose) polymerase (PARP)
• A key regulator of DNA damage repair processes
• Involved in DNA base-excision repair (BER)
• Binds directly to DNA damage
• Produces large branched chains of poly (ADP-ribose)
• Attracts and assists BER repair effectors
XRCC1 Lig3
PNK
Polß
Tutt A on behalf of ICEBERG investigators.
ASCO 2009;Abstract CRA501
Strand Directed Mismatch Repair System
• Removes replication errors not recognized by
replication machine
• Detects distortion in DNA helix
• Methylation occurs shortly after replication occurs
• Reduces error rate 100X
• 3 Step Process recognition of mismatch
excision of segment of DNA containing mismatch
resynthesis of excised fragment
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 Strand Directed Mismatch
Repair in Mammals

• Newly synthesized strand is

preferentially nicked and can be
distinguish in this manner from
parental strand
• Defective copy of mismatch repair
gene predisposed to cancer

Figure 5-20a Molecular Biology of the Cell (© Garland Science 2008)

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 DNA Repair

DNA Repair
►Despite 1000’s of alterations that occur in
DNA each day, few are retained as mutations DNA Damage Can Activate Expression of
►Efficient repair mechanisms Whole Sets of Genes
►Importance of DNA repair highlighted by: • Heat Shock Response
• SOS Response
Number of genes devoted to DNA repair
 mutation rates with inactivation or loss of DNA
repair gene

►Defects in DNA repair associated with

several disease states

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 Failure of DNA repair
• When DNA repair fails, fewer mutations corrected  increase in
number of mutations in the genome.

• The protein p53 monitors repair of damaged DNA.

• If damage too severe, p53 protein promotes programmed cell
death (apoptosis)

• Mutations in genes encoding DNA repair proteins can be inherited

 overall increase in mutations as errors or damage to DNA no
longer repaired efficiently.

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 Protein dynamics to and from sites of DNA breaks.

DNA replication and repair

disorders
Disorder Frequency Defect
Fanconi’s anaemia 1/22,000 in some Deficient excision
popns. repair
Hereditary nonpolyposis 1/200 Deficient mismatch
colon cance repair
Werner’s syndrome 3/1,000,000 Deficient helicase
Xeroderma pigmentosum 1/250,000 Deficient excision
repair

Polo S E , and Jackson S P Genes Dev. 2011;25:409-433

Copyright © 2011 by Cold Spring Harbor Laboratory Press

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 OMIM 278700) XP xeroderma pigmentosum

Caused by homozygosity
For a recessive mutation in
A repair gene.

One example of a DNA-repair genetic disease

Table 5-2 Molecular Biology of the Cell (© Garland Science 2008)

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