Lecture 6

DNA Replication (part II)

1
 DNA Replication
p (p
(part II))

Lecture Outline: Readings:

• Issues in replication
p Alberts textbook,, Ch 5,,
• DNA repair pp. 276-288, 292-304

2
Bacterial DNA replication

3
 A quick key word review
1) Leading strand is synthesized ___________ continuously from
continuously

single
single RNA RNA
______________ primer(s)
2) Lagging strand is synthesized ______________ discontinuously
discontinuously

multiple primer(s)
from _________
RNA primer + DNA
3) Okazaki fragments: _________________
4) DNA synthesis proceeds in which direction?
5’  3’
______
5) Primosome:
Pi h li + primase + primase
helicase
________________
helicase i
6) The predominant helicase is on which strand?
lagging
_______
 Issues in DNA Replication

1) What happens at the ends of eukaryotic

linear chromosomes during replication
2) How is DNA unwound?
3) How are mistakes found and corrected?

5
What happens at the ends of chromosomes?

Alberts, Figure 5-7

6
 Th problem
The bl att ends
d off chromosomes
h
Leading strand:
3’ 5’

5’ 3’
Parental helices are pulled apart ---->

Lagging strand:
5’
5
3’

5’ 3’

Why is the shortening of the 5’ end of the

daughter DNA a potential problem?
Loss
___________
Loss of information
of sequence sequence information
For which strand is this a major problem?
Lagging
________
Lagging 7
 What happens at the ends?
In the majority of eukaryotes,
eukaryotes this problem is
solved by having repetitive DNA sequences at
the ends of the chromosomes. The ends are
called:
hybridise back into the same
 telomeres
telomeres
strand

5’
5 3’
3

3’ 5’

8
 Telomerase to the rescue
The repetitive Telomerase
sequence that is added
to the 3’
3 end of the
parental strand (i.e. the
lagging
gg g strand
template) is determined
by the RNA template in
telomerase.

RNA template DNA comp. copy

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 Telomere Replication

1 RNA template
RNA template

2) Resembles:
Reverse
Reverse transciptase

Transcriptase
3) Generates:
GENERATES:
G-rich ends

G-rich ends
4) Adds
nucleotides
l tid tto:
3’ ends
3' ends
template
of
of lagging strand

parental strand
template 10
 Homeostatic control of telomere length
telomere length is tightly regulated and controlled by the cell

11
Alberts, Figure 5-43
Telomeres and Cancer

Most
• Mostcancer cells produce
cancer cellshigh levels
produce
of telomerase
high
g levels of telomerase
• Modification
Modification of the RNA
of the telomerase
template interferes
telomerase RNAwith cancer cell
template
growth
interferes with cancer cell
growth
Prognoses of someof
• Prognoses cancers
some (neuroblast-
cancers
oma) can be ascertained by telomerase
(
(eg.
levels neuroblastoma)
bl ) can bbe
ascertained by telomerase
levels
• Cell-targeted
Cell-targetted inhibitors
inhibitors of
of telomerase
activity have been suggested as thera-
telomerase activity have been
peutic agents
gg
suggested as therapeutic
p
agents
 Issues in DNA Replication

1) What happens at the ends of

chromosomes?
h ?
2) How is DNA unwound?
3) How are mistakes found and
corrected?

13
 The winding problem

1) Supercoils in same
direction as the ttwist
ist of
the double helix:
 + supercoils
2) Opposite direction:
 - supercoils
3) Replication introduces
supercoils in which
direction?
 + supercoils
Alberts, Figure 5-21 14
Stress released by Topoisomerase type I

1) Type of break:
Single
Single-stranded

stranded
2) This allows DNA
to:
R around
Rotate
Rotate t t the
around d
sugar-phosphate
backbone of one strand
the sugar-PO4
backbone of
one strand

15
Alberts, Figure 5-22
 Topoisomerase type II to untangle and separate

1 Type of break:
1. Double stranded
double-stranded

2. This allows: One ds helix to pass

one double-stranded helix to pass through the other

th
through
h th
the other
th
Alberts, Figure 5-24 16
 Issues in DNA Replication

1) What happens at the ends of

chromosomes?
h ?
2) How is DNA unwound?
3) How are mistakes found and
corrected?

17
The High Fidelity of DNA Replication
1) RNA polymerases typically have an
1 in 104
error rate of about ___________.
1 in 10^4
DNA
polymerases,
l on th
the other
th hand,
h d are
1 in 109
only about _________
1 in 10^9

2) The human genome (3x109) is only

g by
changed 3
y about ______ nucleotides
3. THREE

every time a cell divides!

How is this incredible fidelity maintained?

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 DNA Proofreading

Two separate mechanisms:

3’toto5' 5’ exonuclease
1) 3'
______
Strand-directed mismatch repair
2) _____________
Strand-directed

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Proofreading Exonuclease

This exonuclease:
 chews
Chews back
back the the
misincorporated
nucleotides
mis-incorporated
nucleotide

Alberts Figure 5-8

Alberts,
20
 Switching from Polymerization to Editing for
Proof-Reading

polymerising editing

Figure 5-9 Molecular Biology of the Cell (© Garland Science 2008)

 Why 5’ to 3’
synthesis?

 Only y 5’ toallows
5'-3' direction 3’ for both
efficient error correction and chain
direction allows for
growth

both efficient error

correction and
chain growth
g

Alberts, Figure 5-10

22
 DNA Proofreading

Two separate mechanisms:

3’ to 5’ exonuclease
1) ______
Strand-directed mismatch repair
2) _____________

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 Strand-directed Mismatch Repair
p in Eukaryotes
y

This is a post-
polymerase error
repair
i process
Initiated by detection
of distortion in the
geometry of the
double helix generated
by mismatched
basepairs

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

 Damaged DNA

1) Even after synthesis, DNA can get

damaged and need repair
2) Defects in repair mechanisms have
b
been lilinked
k d tto a variety i t off h human
diseases
 examplesExamples include
include breast, codon, breast, colon, skin
and skin cancers

cancers

25
 DNA can be damaged by:

1) Oxidation
oxidation

2)) radiation
Radiation

3) heat
Heat

4) chemicals
Chemicals

As well as other cell

stressors
MBoC, Figure 5-46 26
 Spontaneous damage to DNA can
also occur
Depurination:
p

Deamination:

27
Alberts, Figure 5-45
 How mutations are produced
p
If uncorrected,
uncorrected mutations now appear in daughter cells

Alberts, Figure 5-47

28
 Base excision repair
This type of
directed repair
targets:
U Uracil  nucleotide
1 nucleotide
Glycosylase

Following the glycosylase,

glycosylase
an endonuclease and
Following removal of phosphodieasterase
remove the
th sugar
Polymerase phosphate
and DNA
ligase 29
Alberts, Figure 5-48a
Nucleotide excision repair
p

This type of
directed repair
targets:
 many nucleotides, excision nuclease
and DNA helicase
Many nucleotides
DNA polymerase
Excision Nuclease and
DNA Helicase

DNA Polymerase and

DNA ligase
DNA Ligase
g
Alberts, Figure 5-48b 30
The End