386
Cell Nucleus Biogenesis, Structure and Function
DNA-binding protein
Swivel
Swivel
Clamp
Lagging
polymerase
RNA primer
RNAse
DNA
Pol
Ligase
Helicase
Primase
Leading
polymerase
3
5
5
3
Fig. 1
Replication proteins of a single replication
fork. Two replication complexes are required to
replicate each replication fork. The activity of the two
complexes is coordinated in the cell – they are thought
to be associated as shown here. The requirement for
leading and lagging strand synthesis is described in
the text. In mammalian cells, the DNA clamp, which
holds the polymerase complex on DNA is a trimer of
PCNA (proliferating cell nuclear antigen) and the
primer that is needed to activate DNA synthesis is a
component of DNA polymerase-
α
. DNA synthesis is
performed by the combined activities of DNA
polymerases-
α
,-
δ
,and-
ε
. The swivel that is required
to release superhelical stress that develops during
elongation is provided by topoisomerases I and II.
(Reproduced from von Hippel, P.H., Jing, D.H. (2000)
Structural biology – Bit players in the trombone
orchestra,
Science
287
, 2435–2436 and adapted from
Yuzhakov, A., Kelman, Z., O’Donnell, M. (1999)
Trading places on DNA – A three-point switch
underlies primer handoff from primase to the
replicative DNA polymerase,
Cell
96
, 153–163. With
permission of American Association for the
Advancement of Science and Cell Press.)
strand can occur continuously in the 5
0
to 3
0
direction. This is called the
lead-
ing strand synthesis
. On the other strand,
called the
lagging strand
, replication oc-
curs only when a single-stranded patch
of about 200 bp has formed. Synthesis
then proceeds to Fll the patch, so that
the replication products on the lagging
strand are all initially short, in mammalian
cells typically 100 to 500 bp in length.
These short fragments, called
Okazaki
fragments
, are then ligated together – by
an enzyme called
DNA ligase
–in a pro-
cess that replaces the RNA primers with
DNA and joins the gaps between adja-
cent Okazaki fragments. It is believed
that the leading and lagging strand en-
zyme complexes are associated so that
their activities can be readily coordinated
(±ig. 1) and it has even been suggested
that the complexes associated with the two
forks arising from each origin are held
together.
2.2.1
DNA Repair
The maintenance of genetic integrity de-
mands that the DNA is duplicated faith-
fully from one cell generation to the next
and that any damage that might arise in
the DNA can be recognized and corrected.
In mammalian cells, our best estimates
suggest that the sequence of DNA changes
at a rate of 1 bp/10
9
bp replicated dur-
ing each division cycle. In fact, it is
important that this level of accuracy is
maintained, as most mutations in protein
coding genes are likely to be deleteri-
ous. Repair is clearly extremely important.
Xeroderma pigmentosum (NER), Blooms
syndrome (ligase) and ataxia telangiectasia
(ATM) are examples of diseases in which
affected individuals have defective DNA re-
pair pathways. These defects correlate with
a massively increased risk of cancer.
In proliferating cells, a major potential
source of DNA mutation is base misincor-
poration during the course of replication.
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