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chapter 24
DNA Replication, Repair, and Mutagenesis
S ite of
V
5‘
missing
f
n u c le o tid e
L ig a s e s e a ls n ic k
L ig a s e c a n n o t s e a l a g a p
....
1
....
I
_________________________________
(a )
N o a c tiv ity
(b )
F I G U R E 2 4 - 4
Action of DNA ligase, (a) A nick having a 3'-OH and a 5'-P terminus
sealed (left panel), (b) If one or more nucleotides is absent, the gap cannot
be sealed.
DNA strands, synthesis of one new strand of DNA can be
continuous but synthesis along the other strand must occur
by
d isco n tin u o u s replication .
At each replication fork one DNA strand has a free
3'-OH group; the other has a free S'-PO^- group. Since
DNA can only elongate in a 5' to 3' direction, syn-
thesis of one strand (the
lea d in g stra n d )
is continuous;
synthesis of the opposite strand (the
la g g in g stra n d )
is
discontinuous.
Short fragments of DNA are synthesized in the repli-
cation fork on the lagging strand in a 5'to 3' direction;
these are called
O k a za k i fra g m e n ts
after the scientist
(Reiji Okazaki) who first demonstrated their existence dur-
ing replication. These Okazaki fragments are joined in the
replication fork by the enzyme DNA ligase that can form
a phosphodiester bond at a single-strand break in DNA.
DNA ligase joins a 3'-OH at the end of one DNA frag-
ment to the 5'-monophosphate of the adjacent fragment
(Figure 24-4). However, if even one nucleotide is missing
in the DNA strand, ligase cannot seal the sugar-phosphate
backbone.
24.2 Enzymology of DNA Replication
The fundamental enzymology of DNA replication derives
from both
in vivo
and
in vitro
studies with cells and ex-
tracts derived from
E. coli.
Many of the enzymes involved
in DNA replication were identified by isolation of condi-
tional lethal mutants of the bacterium, e.g., mutants that
are unable to replicate DNA (and unable to grow) at high
temperatures (42°C) but that replicate and grow normally
at low temperatures (30°C).
The synthesis of DNA is a complex process because of
the need for faithful replication, enzyme specificity, and
topological constraints. Approximately 20 different en-
zymes are utilized in bacteria to replicate DNA. In addi-
tion to polymerization reactions, DNA replication requires
accurate initiation, termination, and proofreading to elim-
inate errors.
DNA Polymerases
Three DNA polymerases have been characterized in
E.
coli
and are designated polymerase I, polymerase II, and
polymerase III (Table 24-2). Although present in very low
concentration in the cell, polymerase III, also called
repli-
case,
is the polymerase that elongates both strands of the
bacterial DNA in the replication fork. Polymerase I is pri-
marily a DNA repair enzyme and is responsible for exci-
sion of the short RNA primer that is required to initiate
DNA synthesis on both the leading and lagging strands of
DNA during replication. It also can remove mismatched
base pairs during replication and fill in gaps in single-
stranded DNA that is joined in a double helix. The function
of polymerase II is not clear but it probably also has repair
functions.
All DNA polymerases select the nucleotide that is
to be added to the 3-OH end of the growing chain
T A B L E 2 4 -2
P ro p erties o f
E.
c o li
DNA
p o lym era ses
Property
Polymerase
I
II
Ill
Molecular weight
105,000
90,000
130,000
Molecules/cell
-400
-100
-10
Nucleotides/sec
-20
-5
-1,000
3' exonuclease activity
yes
yes
no
5' exonuclease activity
yes
no
no
Biological activity
RNA primer excision, DNA repair
SOS DNA repair?
Replicase
