The Replication of DNA

CHAPTER 8

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

multiple DnaA-ATP proteins bind to the repeated 9-mer 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

Take E.coli for example

DNA helicase

DNA helicases unwind the double helix

Replication in details

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 .

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

SHORT RNA

PRIMERS

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 molecul

es How to copy the extreme ends of the la

ging strand

Two solutions !!!

Type topoisomerases are required to separaⅡted daughter DNA

After a circular DNA molecular is replicated , the resulting complete daughter DNA molecules remain linked to one another . Type topoisomerases can sepⅡatate 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 )

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

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

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 initiate the replication

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 .