Lecture 6

DNA Replication (part II)

Bacterial DNA replication

Aq
quick key
y word review
continuously
1) Leading strand is synthesized ___________
continuously from
single
RNA
single
RNA
______________
primer(s)
discontinuously
discontinuouslyy
2)) Lagging
gg g strand is synthesized
y
______________
multiple primer(s)
from _________
RNA p
primer + DNA
3)) Okazaki fragments:
g
_________________
4) DNA synthesis proceeds in which direction?
5 3
______
+ primase
helicase
+ primase
5) Primosome: helicase
________________
6) The predominant helicase is on which strand?
lagging
_______

DNA Replication (part II)

Lecture Outline:

Readings:

Issues in replication
DNA repair

Alberts textbook, Ch 5,
pp 276-288
pp.
276 288, 292-304
292 304

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?

What happens at the ends of chromosomes?

Alberts, Figure 5-7

The problem at ends of chromosomes

Leading strand:
5

3
5

parental helices pulled apart this direction --->

Lagging strand:
3
5

Why is the shortening of the 5 end of the

d
daughter
h DNA a potential
i l problem?
bl ?
loss
of sequence
Loss
ofinformation
sequence information
___________
For which strand is this a problem?
lagging
strand
Lagging
________

What happens at the ends?

In the majority of eukaryotes, this problem is
solved
l db
by h
having
i repetitive
titi DNA sequences att
the ends of the chromosomes. The ends are
called:
telomeres
telomeres
5

Telomerase to the rescue

The repetitive
p
sequence that is added
to the 3 end of the
parental strand (i
(i.e.
e the
lagging strand
template) is determined
by the RNA template in
telomerase.

Telomerase

RNA template --> DNA complementary copy

RNA template DNA comp. copy

9

Telomere Replication
Telomerase:
1 RNA template
2) Resembles:
Reverse
Reverse
transcriptase
Transcriptase

3) Prefers G-rich
ends G-rich
4) Adds
nucleotides to
3endsends
3
of
3'
of lagging
strand template
lagging strand
template

10

Homeostatic control of telomere length

g

11

Alberts, Figure 5-43

Issues in DNA Replication

1) What happens at the ends of
chromosomes?
2) How is DNA unwound?
3) H
How are mistakes
i t k ffound
d and
d
corrected?

12

The winding problem

1) Supercoils in same
direction as the twist of
th d
the
double
bl h
helix:
li
+ supercoils
2) Opposite direction:
- supercoils
3)) Replication
p
introduces
supercoils in which
direction?
+ supercoils
Alberts, Figure 5-21

13

Stress released by Topoisomerase type I

1) Type of break:
Single
single
stranded
stranded
2) This allows DNA
t
to:
rotate
around
Rotate
around
sugar-Po4
backbone
oneg
theofsugar-PO
4
strand, requires no
ATPbackbone of
one strand

Alberts, Figure 5-22

14

Topoisomerase type II to untangle and separate

protein gate

1. Type of break:
2. This allows:
Alberts, Figure 5-24

Double stranded

double stranded

pass
One ds helix to p
through the other

one double-stranded helix to pass through the other

15

Issues in DNA Replication

1) What happens at the ends of
chromosomes?
2) How is DNA unwound?
3) H
How are mistakes
i t k ffound
d and
d
corrected?

16

The High Fidelity of DNA Replication

1)) RNA p
polymerases
y
typically
yp
y have an
error rate of about ___________.
DNA
1 in 104
polymerases, on the other hand, are
1 iin 109
only about _________
2) The human genome (3x109) is only
3
changed by about ______
nucleotides
every time a cell divides!
1 in 10^4

1 in 10^9

How is this incredible fidelity maintained?

17

DNA Proofreading
Two separate mechanisms:
3 to 5 e
1)) ______
exonuclease
o uc ease
Strand-directed mismatch repair
2) _____________
3' to 5'

strand-directed

18

Proofreading Exonuclease

polymerase needs
last base to be
base-paired right,
and 3' Oh to add
onto

This exonuclease:
Chews
back the back the
chews
mis-incorporated
nucleotide
mis-incorporated
p
nucleotide

Alberts, Figure 5-8

19

Switching from Polymerization to Editing for

P f R di
Proof-Reading

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

Why 5 to 3
synthesis?
only 5
Only
5 to 3
to 3
direction allows for
both efficient error
correction and
chain growth

direction allows for

both efficient error
correction and
chain growth

Alberts, Figure 5-10

21

DNA Proofreading
Two separate mechanisms:
3 to 5 e
1)) ______
exonuclease
o uc ease
Strand-directed mismatch repair
2) _____________

22

Strand-directed Mismatch Repair in Eukaryotes

This is a postpolymerase error
repair process
Initiated by detection
of distortion in the
geometry of the
double helix generated
by mismatched
basepairs
prokaryotes, look
for methylation,
eukaryotes looks
for these nicks
Figure 5-20a Molecular Biology of the Cell ( Garland Science 2008)

Damaged DNA
1) Even after synthesis
synthesis, DNA can get
damaged and need repair
2) Defects
D f t in
i repair
i mechanisms
h i
h
have
been linked to a variety of human
diseases
di

Examples include breast, colon, skin

cancers
eg breast, colon,
skin cancer

24

DNA can be damaged by:

1) oxidation
oxidation

2) radiation
radiation

3) heat
heat

4) chemicals
chemicals

As well as other things

Alberts, Figure 5-46

25

Spontaneous damage to DNA can

also occur
Depurination:

depurination

Deamination:

deamination

26

Alberts, Figure 5-45

How mutations are produced

cell has until next replication event to locate and
If ifuncorrected,
mutations
now appear in daughter cells
uncorrected,
correct errors
mutations then
appear in daughter
cells

Alberts, Figure 5-47

27

Base excision repair

Glycosylase

This type of
directed repair
targets:
1 nucleotide
1 nucleotide

removes specific base

Following
g removal of

Polymerase
and DNA
ligase

Following the glycosylase,

an endonuclease and
phosphodieasterase
remove the sugar
phosphate

Alberts, Figure 5-48a

28

Nucleotide excision repair

This type of
directed repair
targets:
many nucleotides
Many nucleotides
Excision Nuclease and
DNA Helicase

Alberts, Figure 5-48b

29

The End