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Differ models of DNA replication

Semiconservative- parental strands go to one parental strand and one daughter strand

Conservative- one helix full parental, one full daughter

Dispersive- a mix of both



E Coli cells were grown in medium with heavy nitrogen 15 as the only source of nitrogen for many generations

Cells transferred to medium containing nitrogen 14. After cells divided, sample was collected and the DNA purified

Cells divided second time, sample collected and DNA purified

Centrifuge the three samples.and compare the location of the bands


Results of meleson and stahl's experiment

Fist gen- heavy band

Second gen- heavy and light

Third gen- mostly light



Combo of fluorescent dye and giesma stain distinguishes between chromosomes with one strand containing BrdU and those with two stands of BrdU


DNA replication

Catalyzed by DNA polymerases, none of which can initiate DNA chains in either direction, can't unwind DNA

Direction of synthesis is 5' to 3'

Both strands are duplicated


What is needed to start DNA replication?

Need Available 3' hydroxyl group for base to come in on- 3' OH attacks phosphate on incoming nucleoside triphosphate

Initiation requires a primer with a 3' OH, Primer made from RNA because DNA polymerase a cannot add on bases by themselves

Many accessory proteins required


Replication forks

Replication forks are where there is unwinding of the parental DNA and the nucleotides are being incorporated into new complimentary strands


DNA replication in prokaryotes

Replication is bidirectional

One replicon


Where does replication begin? (Pro)

Begins at a specific point called the origin oriC

Proteins (DnaA-ATP) bind the origin and initiate replication (actually 30 proteins involved)


Prokaryotic replisome

In vivo, it's thought that DNA polymerase III (holoenzyme dimer), the primosome, and DNA helicases are associated in a replisome that synthesizes DNA at 900bp per second


Initiation (pro)

OriC contains four 9bp binding sites for the initiator protein DnaA

Eventually it will form a group of 30-40 molecules each bound to ATP (negatively supercoiled)

Three 13 bp AT regions regions "melt" open and that allows DnaB (helicase)to bind. Helicases use ATP to move into and "melt" open the double stranded DNA


Separating the DNA strands (pro)

Causes many problems with the topology- DNA gets overwound, so there's positive supercoiling in the unreplicated portion of the DNA

DNA gyrase is a topoisomerase (II) that helps relieve the tension caused by supercoiling further from the origin


DNA Topoisomerase

Type one- Nick one strand of DNA, let it roll, then reattach. Relieves strain.

Type two- Cuts both strands.

Binds one strand of the replicated DNA, close around it and cut it, and next strand will be passed through, then seal the cut.


Unwinding (pro)

DNA helicases must move along the template strands to open it for copying

Other proteins (single stranded binding proteins) also promote further unwinding by stabilizing the single stranded unwound DNA

Positive supercoiling is relaxed by DNA gyrase


Elongation overview (pro)

Two strands of DNA are anti-parallel, replication is anti parallel

One strand, the leading strand, is replicated from 5'-3', one primer

Lagging strand replicated in 3'-5' but it is done in small fragments from 5' to 3' that are joined together as a new fragment is begun at the replication fork by DNA ligase

Okazaki fragments begin with RNA primer put down by enzyme primase


Elongation steps (pro)

Primase and helicases form a primosome. Periodic binding of the primase gives short RNA primers that generate the Okazaki fragment

Both strands elongated by DNA polymeraseIII

Lagging strand primers are removed and the gaps are filled by DNA polymerase I

Final phosphodiester bond between fragments is formed by DNA ligase- joins fragments so there is a continuous strand of DNA on the lagging strand


DNA polymerase III

This multiunit holoenzyme is a dimer- all subunits are required for it to work

One half synthesizes the leading strand and one half synthesizes the lagging strand

Two polymerases in the same strand is good- Speed efficiency, lagging and leading at the same time


What does each half of the dimer of DNA polymerase III have?

Both halves of the dimer have:
An alpha unit (actual polymerase)
an epsilon unit (3'-5') proofreading exonuclease, Can degrade from the edge in. Exo- comes from the outside
Beta subunits clamp the polymerase to the DNA
Other subunits in either half may allow elongation of short or long strands of DNA


DNA polymerase I

Has many exonuclease activities

Removes RNA primers and fills the gaps with DNA

Recognizes mismatched bases


Induced fit

Removal of mismatched base by DNA polymerase I

New strand that just had a base added gets put towards the palm and exonuclease activity cuts the base

Fingers move towards the palm and results in catalysis and incorporation of base, but incorrect incorporation leads to a single stranded 3' end,conformation change leads base to exonuclease


Termination (pro)

Both replication forks meet 180 degrees from the origin

Several sites that stop movement of the replication forks by binding the tus gene product

Topoisomerase IV (type II DNA isomerase) unlinks the two daughter chromosome- both strands cut and rejoined


Eukaryotic DNA replication

Replication occurs during S1 phase of mitosis

Replication forks move at 50bp pr second, would take 30 days to complete

Solved by more origins of replication


Experimental systems

Yeast has a smaller genome 14000kb in 16 chromosomes) and 400 replicons

Viruses, SV40, 5kb double stranded circular genome- good ex of eukaryotic replication fork

Cell free extracts from African clawed frog - can support the replication of added DNA or whole nuclei


Origins of replication (euk)

Replicate the genome in small portions termed replicons

Clusters of about 20-50 replicons begin at the same time throughout s phase- time event coordinated by cell signals

In early s phase it's primarily euchromatin(not compacted) that is replicated


Initiation (euk)

Autonomous replicating sequences (ARSs) - sequences that promote replication

Minimum of 11 bp w/ following sequence: A/TTTTATA/GTTTA/T to support replication- recognition site for origin recognition complex (ORC), recognizes specific hydrogen bond donors and acceptors

2 forks with different directions


Liscencing factors

Liscencing factors are required for initiation then inactivated after use. Can only get into nucleus when nuclear envelope disappears



Beginning of s-phase, kinases activated- CDK is a cyclin-dep kinase which has high activity in s phase and all mitosis. Suppresses the formation of new per replication complexes so that each origin can only be activated once per cell cycle


Pol alpha

associated with primase and helps with initiation of Okazaki fragments


Pol beta

DNA repair


Pol delta

primary DNA synthesizing enzyme during replication, requires a sliding clamp to maintain association (PCNA)


Pol epsilon

works. With pol delta in synthesizing DNA


Pol gamma

replicates mtDNA


Polymerase delta-PCNA-rfc complex

Polymerase delta-PCNA-rfc complex replaces primase-polymerase alpha complex and extends the short primer, generating the leading strand

As the helicase further unwinds the parental strands, the primase-polymerase alpha complex makes a primer for the lagging strand, and polymerase delta-PCNA-rfc complex synthesize the Okazaki fragment


primase-polymerase alpha complex

Primer removed by RnaseH and FENI and polymerase delta fills the gap


Histones during replication

DNA must be unwound from the nucleosomes

As the fork passes new nucleosomes are formed from both old and new histones

Histones are added to the lagging strand after DNA ligase has sealed the backbone

CAF1 associates with PCNA to bring in histone proteins. Between 180 and 210 start winding on histones.


Elongation (euk)

Helicases unwind the DNA, single stranded binding proteins (RP-A)

Three polymerases involved in elongation:
-alpha- primase FHA helps with the RNA primer that begins elongation on the leading and Okazaki fragments on the lagging strand
-delta- replaces alpha and continues elongation, the ability to synthesize long fragments of DNA is aided by proliferating cell nuclear antigen (PCNA)
-epsilon- maybe involved in completing lagging strand fragments
(Delta and epsilon have proof reading capability)


What is replicated last in eukaryotes?

Centromeres and telomeres are replicated last

Heavy in satellite repeats


Chromosome shortening

Helicase unwinds end of DNA helix (At end of chromosome)

DNA polymerase completes the leading strand. Primase synthesizes RNA primer at the end of the lagging strand

DNA polymerase synthesizes the last Okazaki fragment in lagging strand

No DNA synthesis occurs after primer is removed (no free 3' end for DNA polymerase); chromosome is shortened

Bases available at the end could cause recombination events. Also kind of sticky, so you would have chromosomes that would fuse together because they have unreplicated ends


Why is telomere replication different from the rest of DNA?

Last areas of DNA to be replicated

Not replicated in the same way as the rest of the chromosome- Not enough DNA exposed to generate an Okazaki fragment

Consist of tandem repeat sequence (TTAGGG-human) with the 3' end overhanging the 5' end


Telomerase (reverse transcriptase) activity

Strand of RNA is complimentary to the repeat motif of the satellite found in the telomeres. Adds DNA bases to RNA compliment with reverse transcriptase

Elongation, translocation, elongation


Telomere replication

When RNA primer is removed from the 5' end of the lagging strand, a stand of parent DNA remains unreplicated

Telomerase binds to the overhanging section of single stranded DNA. Telomerase adds deoxyribonucleotides to the end of the parent DNA, extending it

Telomerase moves down the DNA strand and adds additional repeats

Primase, DNA polymerase, and ligase then synthesize the lagging strand in the 5'-3' direction, restoring end original length of the chromosome


Why are short chromosomes bad?

Shorter chromosomes are a signal for apoptosis

Cancer cells has mad skills at telomerase, no cell death due to no chromosome shortening


What about the overhang?

RNA primer at the 5' end will be removed but a gap remains

This single stranded overhang will fold into a t loop around specialized proteins to protect the end of the DNA molecule

Loop seals off the end of the chromosome so the overhang doesn't try to base pair with anything


Sheltering complex

POT1 plus TRF1 and2. If there is a high concentration of POT1, to inhibits telomerase and it means that the chromosome is of adequate length.