Adenovirus and some bacterial viruses do not make Okazaki fragments'

1
Adenovirus and some bacterial viruses do not make
Okazaki fragments.
The primer for DNA synthesis of these viral
genomes is a protein-pA-3-OH complex
2
Replication in most cells/viruses is
bi-directional (some plasmids replicate
unidirectionally). Two replication forks move
away from each other from each origin or
replication. The two forks do not always start
at the same time.
Bidirectional replication
Unidirectional replication
Fig. 20.11
Fig. 20.10
A replicating circular chromosome using a single
origin reveals a theta structure
Fig. 20.9
3
Fig. 20.14
Phage l replicates initially by ? replication and
then by a rolling circle or s replication

• replication is bi-directional?
• s replication is unidirectional

Fig. 20.13
Some viruses and plasmids replicate by a rolling
circle mechanism
4
Mutants that are Ts for DNA synthesis are
classified as fast- or slow-stop.
Slow-stop mutants may be defective only in the
initiation phase of DNA replication, e.g., dnaA,
dnaC. But dnaL (DNA ligase) has a slow-stop
phenotype, as may leaky elongation mutants
Fast-stop mutants are defective in the elongation
phase of replication, e.g., dnaB, dnaE, dnaG,
ssb, gyrA
dnaB(Ts)
dnaA(Ts)
Fig. 20.20
5
There are 5 DNA polymerases in E. coli. Pol III
is the replicating enzyme. Only Pol III is
absolutely essential for cell growth.
All DNA pols are involved in aspects of repair of
DNA damage.
Pol I and Pol II have intrinsic exonuclease
proofreading activities, Pol III has a tightly
associated exonuclease.
Pol IV and Pol V lack proofreading activities and
are mutagenic enzymes. They are used primarily
when damage is too severe for error-free repair
pathways
dnaE
dnaQ mutD
dnaX
dnaZ
dnaN
6
Synthesis by Pol III (in the presence of SSB or a
helicase) is very processive
The PAS allows binding of the pre-primosome, a
protein complex that normally loads primase onto
DNA. Pre-primosomes have 5-gt3 helicase activity
Pol III HE SSB OR Pol III HE helicase
Fig. 21.8
5
Pre-primosomal proteins SSB
DnaB (5-gt3 helicase)
No primase was present in these expts to initiate
synthesis on the lagging strand. Addition of
primase could have made the long ssDNA tail into
dsDNA
The rate of DNA synthesis, 0.75 kb/sec at 37C,
is close to the in vivo rate
Fig 21.9
7
There are now more than a dozen mammalian DNA
polymerases, most are specialized DNA repair
enzymes. Replication is carried out by the
combination of DNA polymerase a and d, together
with associated replication factors
DNAP ? is essential in yeast
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9
Replication functions are generally conserved
across kingdoms
10
DNA replication requires many accessory factors
in addition to DNA Pol, including primase, DNA
helicase(s), single-stranded DNA-binding proteins
(SSB), a clamp that makes replicative DNA
synthesis processive, and topisomerases
SSBs (early terms include DNA-unwinding proteins,
helix-destabilizing proteins) are not enzymes,
they bind stoichiometrically and cooperatively to
ssDNA. They protect the more reactive ssDNA from
damage, and by binding to other replication
proteins they help the formation and stability of
the DNA replication machine.
The eukaryotic equivalent of SSB is called RPA
(Replication protein A). The human protein is
required for multiple processes in cellular DNA
metabolism including DNA replication, DNA repair
and recombination. RPA is composed of three
tightly associated subunits of 70, 32, and 14
kDa. All three subunits of RPA are required for
function however, only the 70 kDa subunit has
high-affinity SSB activity. In addition to
binding to ssDNA, RPA also interacts with
multiple proteins. These specific interactions
are important for RPA function. In DNA
replication RPA-protein interactions appear to be
essential for initiation of DNA replication and
for efficient movement of the replication fork.
RPA becomes phosphorylated at the beginning of
S-phase and after cellular DNA damage. Changes in
the phosphorylation state of RPA may be important
for coordinating cellular DNA metabolism.
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14
The E. coli and yeast DNA Pol sliding clamps are
similar in structure
Yeast PCNA
E. coli ?
Fig. 21.12
Fig. 21.14
15
In E. coli the clamp loader is called the ?
complex, in eukaryotes it is called RFC,
replication factor C. ? and RFC are structurally
and functionally similar
16
The replicating DNA Pol holoenzyme has two active
sites for DNA synthesis
Fig. 21.17
Why?
17
Trends Microbiol, 2007
18
Fig. 21.19
The lagging strand DNA Pol recycles via its
affinity to the b sliding clamp
A dimeric DNA Pol can replicate both strands at
the same time. One synthesizes the leading
strand, the other the lagging strand
Fig. 21.18
Fig. 21.20