L3 - DNA replication

On a DNA melting curve what is Tm

Tm= half DNA is single stranded and half DNA double stranded

• the higher the Tm value the more G-C bonds you have as have more H bonds

DNA replictaion is ( 2 things)

• semi-conservative (half parental half newly synthesized stand)

• happen bidirectionally

where does replication start

At the point of origin

DNA replictaion ends at

termination site

what is replicosome

all the machinery involved in DNA replication

how many replication forks are there

2 as replication is bidorectional

where replication begins in prokaryotes

• 1 orgin of replication called OricC site (euk have many orgins)

• form 1 replicon (as1 site of origin)

• have termination sites

DNA replication in Euk

• have multiple sites of orgin

• not all orgins of replication are activated at the same time (way to regulate DNA replication)

• form multiple repicons due to many sites of orgin

• picture shows how it works

Replication model for replication initiation in prok..

• initiator DNA binding proteins Recognize OriC site

• downstream have 5 regions of 9 mer and 3 regions of 13 mers

• these are also recognized by specific DNA binding proteins to then start DNA replication

What are the DNA binding proteins involved

DnaA

DnaB

DnaC

what does DnaA do?

• DnaA binds to the 5 9mer regions (each 9mer bound with 1 DnaA = so 5 DnaA bound)

• DnaA has ATP bound which interacts with 13mer regions to open up DNA creating replication bubble

what does DnaB do ?

• it is a helicase

• a hexametric protein and has a ring structure

• it is inactive when bound to helicase loader

what id DnaC?

• it is helicase loader helps helicase attach to DNA

Intercation between DnaB and DnaC

• once bound to DNA helicase is still inactive becuase still bound to helicase

• primase synthesis a short RNA primer at BOTH replication forks which causes helicase loader to dissociate and so activating helicase

what are SSB’s

single stranded DNA binding proteins which keep DNA open - stop it from reanealing back together

bind to single stranded Dna after helicase has opened it

what ahppens after primer formation form primase enzyme

• RNA primer formed at BOTH replication forks get helicase loader to dissociate

• DNA polymerase to come and attach to primers (to the 3’ hydroxyl group)

• have 2 DNA polymerases moving towards replication fork while DNA helicase still working

types of tropoiomerases

type I —> nicks one stand of DNA, relasing a few of those stands then rejoing it

type II —> makes break in both double stands of DNA and rejoins them after

• they stop DNA for becoming too coiled

difference between positive and negative super coiling

• negative —> that region of DNA is opened, so less coils there

• positive —> more coils and turns in that region

At end of DNA replication what happens to that RNA primer

• gets degraded and replaced by DNA joined to strand using DNA ligase

Formation of the phophodiester bond

• the complementary base pair gets close enough to stand so that

• 3’ Hydroxyl group initiates a nucleophilic attack on the phosphate group (alpha phosphate) of the complementary base pair

• this forms a phosphodiester bond

• remaining 2 phophates are called pyrophosphates and are broken down by pyrophosphotase which releases energy for this whole recation to take place

• usually non comp bases do not join together as do not get close enough for nucleophilic attack to happen

how does synthesis of new stand actually happen

• Synthesis occurs from 5’ - 3’ direction

• on the leading stand synthesis easily happens

• as lagging strand is anti-parallel harder for this to occur

• what happens is small RNA primer binds and then get small epice of DNA syntheises in 5’ -3’ diection

• RNA primer then detached and moves backwards and retached then get DNA synthesie form 5’ to 3’

• this contously happens fomring olkazaki fragments which are joined using different DNA polymerase 1 (in prokaryotes)

• there are 3 parts to DNA polymerase one used in leading stand other two used in lagging strand

what problem occurs in prok after DNA replication and how is this solved ?

• Circular DNA will be joined together

• in order to separate them tropoiosomerase 2 makes a cut in one DNA and loops it out of the other

• then have 2 separate circular DNA

how DNA polymerase works

• shaped like a hand

• the palm of hand is the active site for polymerization reaction

• if incorrect base is detected hand closes and catalytic activity is paused

• Incorrect base moved ot exonuclease domain to then be removed (3’ to 5’ exonuclease activity)

• then hand relaxes and DNA moves back to polymerase domain when recation is unoaused and picks up speed

why do mistakes not happen often?

• complementary base pairing

• DNA polymerase proof reading

• post replication repair system (Miss-match repair)

What are the steps in PCR

• Denaturing

• Annealing

• Elongation/ Extension