Download The Replication of DNA

CHAPTER
8
The Replication of DNA
Initiation of DNA replication
Replicon : a particular origin that all
the DNA is replicated from
Replicator : the entire set of cis-acting
DNA sequences that is sufficient to
direct the inition of DNA replication ,
including initiator binding sites and
easily unwound DNA
Initiator protein : the DNA-binding
protein specially recognizes a DNA
element in the replicator and activates
the initiation of replication
The easily
unwound DNA is
always a stretch
of AT-rich DNA
Initiator proteins three functions
These proteins bind a specific DNA
sequence within the replicator
 Once bound to the DNA ,they frequently
distort or unwind aregion of DNA adjacent
to their binding sites
 Initiator proteins interact with additional
factors required for replication initiaton

Examples :
The E.coli initiator protein ,Dna A have
three different functions above .
 In eukaryotic cells ,the initiator is a six
protein complx called the origin reconnition
complx (ORC) . But ORC performs two of
the three fuctions : binding and recriting
other replication proteins to the replicator .

Prorein-protein and protein-DNA
interactions direct the initiation process
Take E.coli for example
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multiple DnaA-ATP proteins bind to the repeated 9mer sequences within ORC
binding of DnaA-ATP to these sequences leads to
strand separation within the 13-mer repeats
DNA helicases(DnaB) and the DNA helicases
loader(Dnac) interactions with DonA
DNA helicases loaders catalyze the opening of DNA
helicases protein ring and placement of the ring
around the ssDNA at the origin
DNA helicases each recruit a DNA primase which
synthesizes an RNA primer on each template
Replication in details
DNA helicases unwind the double helix
DNA
helicas
e
The formation of replication fork and
SSBs bind to stabilise ssDNA
Tw decreases as DNA unwinds .
Wr increases as Tw decreases .
Topoisomerases remove positive supercoils .
SHORT
RNA
PRIMERS
DNA replication requires a RNA primer
Primase make short RNA primers using
ssDNA as template
DNA primase is activated by interacting
with the DNA helicase
PRIMASE
DNA polymerases
catalyzes DNA
synthesis
Sliding DNA
clamps increases
processivity
SLIDING DNA
CLAMPS
Processivity : the ability of
an enzyme to catalyze many
reactions before releasing
its substrate
RNAse degrades RNA
base paired with DNA
Removal of RNA primers
leaves gaps
DNA polymerase fill the
gaps
DNA ligase repairs the
remaining nicks
Finishing replication
Questions ?
How to separate daughter DNA
molecules
 How to copy the extreme ends of the
laging strand

Two solutions !!!
Type Ⅱ topoisomerases are required to
separated daughter DNA
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After a circular DNA molecular is replicated , the
resulting complete daughter DNA molecules remain
linked to one another . Type Ⅱ topoisomerases can
sepatate these DNA circles
There is no inherent toplogical linkage after the
replication of a linear molecule , the large size of the
eukaryotic chromsomes necessitates the intrcate
folding of the DNA into loops attached to a protein
scaffold , and these loops must separated by
topoisomerases .
One solution to ens replication problem
Using a protein as aprimer for the last
Okazaki fragment at each end of the
chromosome . In this situation , the
“ priming protein “ binds to the
lagging strand template and uses an
amino acid to provide an OH that
replaces the 3’OH normally provided
by an RNA primer
The other solution ( in most eukarotic
cells )
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Telomerase uses its RNA component to anneal
to the 3’end of the ssDNA regin of the
Telomerase
Telomerase uses its reverse transcription activity
to synthesize DNA to the end of RNA template
Telomerase then displaces the RNA from the
DNA product and rebinds at the end of the
telomerase andrepeats the process
The telomerase regulation
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The proteins bound to the double-stranded
regions of the telomere regulate the tolemere
lengh
As few telomere sequence repeat , few of these
proteins will be bound to the telomere and
telomerase activity will be activated
As the telomere gets longer ,these proteins will
accmulate and inhibit the telomerase
Eukaryotic chromosomes are replicated
exactly once per cell cycle
Pre-RC initiate the replication
The pr-RC formation
The assembly of the pre-rc is an ordered process
that is initiated by the association of the origin
recognition complex with replicator
Once bound to the replicator , ORC recruits at
least two additional proteins , cdc6 and cdt1 .
These three proteins function together to recrit
the putative eukaryotic DNA helicase .
Pre-RC regulated to allowonly a single
round of replication during each cell cycle
Pre-RC’s formation and activation is regulated by
the Cdks
Active Cdk is absent during G1 , but present
during s , G2 and M phases.
There is only one opportunity for pre-RC to form
and one opportunity to activated
Pre-RCs are disassembled after they are activated
or after the DNA to which they are bound is
replicated .