DNA Replication Flashcards
Meselson and Stahl (1958)
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
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
Results of meleson and stahl’s experiment
Fist gen- heavy band
Second gen- heavy and light
Third gen- mostly light
BrdU
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
CDK
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.
