DNA Replication Flashcards
Mendelson + Stahl (Overall)
“most beautiful experiment in biology” – shows semi-conservative replication
How does DNA replicate (overall)
Replicates in semi-conservative fashion
dsDNA seperates –> new DNA is synthesized onto each template
After synthesis – each dsDNA has one strand of newly sythesized DNA
Imortance of DNA replication
Essential for life – basic mechanisms are shared in all living organisms
Bacteria vs. Euk replication
Main differnece = bacteria is circular genomes = need mechanism to make closed circle of DNA Vs. Euk have linear chromsomes (have different mechanims to replicate linear telemere sequance at the end of chromsomes)
Replication phases
3 Phases
- Initiation –> Unwind DNA + Find starting point + build replication form
- Elongation –> DNA is replicated
- Termination –> Finish replication
Bacteria Start of replication
Bacteria – DNA is compressed through supercoiling –> topisomerases enzyme that over/under wind DNA
Intiation
Gyrase
Decide where to start –> once unwind = can access DNA –> ORI SITE
DNA A –> Get ssDNA
Biuld replication form – Helicase
- DNA B (Hleicase) = loaded to ss of DNA with help of DNA C (DNA helicase loader)
- Helicased = loaded onto each ddDNA
Gyrase
A specific Topisomerase that unwinds supercoild DNA for DNA replications
- Removes + supercoiling
How does Gyrase bind to DNA
To unwind – Gyrase = binds to sequens of DNA –> cuts through dsDNA and unwinds one loop = shifts DNA molecule from one domain of the enzyme to another domain of enzyme –> then gyrase glues ends back
Requires ATP
What does Gyrase require
Requires energy in the form of ATP
What inhibits Gyrase
Classes of Antibiotics “quinoloes” = prevents bacteria DNA replication by targeting ATP binding sites of Gyrase
Ex. Naladixic acid
Origin of replication
Region of DNA that is origin of replication = ori site
Bacteria = only one ori site on circular chromosome
Ori site
Origin of replication
Conatins 3 replication regions that have high number of A/Ts –> AT rich region = followed by 9 BP seq
9 BP seq = DNA A box
Intersperesed through 275 ori sequence = repeats of 4 BP GATC
DNA A box
9 BP sequence after Ori site
repeats 5 times in E.coli
DNA A recogniztion sequence
GATC sequence – C must be methylated
GATC in Ori
Interspersed in ori sequence = repeats of GATC
For DAN replication to occur C in GATC = chemocally odifies through methy group
C = methylated on both strands –> methyl groups help porteins reconnize ori sequnce
DNA A
DNA binding protein that scans genome looking for DNA A box –> When DNA A binds to DNA box = causes dsDNA to bend
- DNA A causes tension to DNA – H bonds are broken in AT rich region of Ori sequence = seperate dsDNA
When many DNA A proteins are bound to ori site = get bending of helix structure = creates physical stress –. To releive stress – H bonds between A-T base pairs and AT sequnces in DNA A box are broken = dsDNA seperates = get ssDNA to act as tenmplate for replication
Helicase
DNA B – Multimerix complex shapes like donut
To get around ssDNA DNA C binds to DNA A and to Helicase –> Seperates Helicase ring –> wraps the helicase around DNA and then reforms donut ring
DNA B = seperates dsDNA (unzips 5’ - 3’ to get more ssDNA for replication
- Unwinds replication form
- Creates tension in dsDNA
DNA C
DNA helicase loader
Movement of Helicase
Moves down ssDNA 5’ –> 3’ so each helicase moves in opposite directions
Movement of Helicase = dislodges DNA and DNA C from ori sequnece because DNA C is not needed once the Helicase is loaded
Replication Bubble
Structure created in intiation
Conatins 2 replication forks
Replication forks
Site where ssDNA is being seperated
Stability of ssDNA + ssDNA binding proteins
ssDNA is NOT stable – will try to form dsDNA –> To prevent dsDNA from reforming ssDNA binding proteins bind non-specifically to ssDNA and prevent H-bonds from reforming between 2 ssDNA
- Allows bubble to get bigger + DNA helicase to do its jobs
Gyrase + Helicase
DNA Gyrase = unqinds the supercoiled DNA caused by helicase
Electron micropscope images of DNA replication
Shows – chrmosomes activley replicating
Shows:
1. Two replication forks in bubbles
2. In bubble have new DNA synthesized from template DNA
Elongation
DNA replicates during Elongation
Overall – Primer binds + replication assmebled
DNA polymerase
Primase –> Get RNA primer
Onece primer is formed = DNA polymerase is recurted
DNA polymerase creates new strands of DNA
Reprisome holds DNA polymerase to allow new DNA to be synthesized
End = close gaps in okazaki fragments
Enzyme responsible for DNA replication
DNA polymerase
DNA polymerase
Enzyme responsible for DNA replication
Binds to ssDNA –> Wraps around DNA
Main function = add free nucleotides to 3’OH end of DNA chain (only add to 3’ end = DNA replication occurs 5’ - 3’)
- Continue adding completely nucleotides (complementary to template)
DNA pol binding to DNA
Binds loossley because needs to move down DNA
Limitation of DNA polymerase
Only adds nulceotides to existing chain – can’t begin DNA replication from scratch
Because can’t begin without free 3’ end of nucleotide chains to build = the first steo in elongation requires sequnece of startong oligionucelotiodes = “primer”
DNA pol in E.coli
5 types of DNA polymerase – main type is DNA polymerase 3
Start of elongation
Because can’t begin without free 3’ end of nucleotide chains to build = the first steo in elongation requires sequnece of startong oligionucelotiodes = “primer”
Primase = Synthesizes RNA primer
Primase
Synthesizes RNA primer –> Can intiate RNA synthesis without existing oligionucelotide strand
Binds to DNA helicase and scnas replication bubble until finds specific target sequnce on ssDNA
When primase finds target –> Uses it as a template for RNA primer synthesis
Primasome
Helicase + Primase complex
How does DNA polymerase hold onto template
DNA polymerase holds onto template DNA by sliding clamp + Sliding clamp loader
Sliding Clamp + Sliding clamp loader
Holds DNA pol. onto template
- DNA polymerase is loaded onto ssDNA by sliding clamp and sliding clamp loader
tethers 2 DNA polymerase on each strand –> Ensures replication occurs on each strand simultaneously
Replisome
Complex of Helicase + Primase + DNA pol + Sliding clamp + Loader
DNA replication occurs at replisome
Creating replisome – DNA polymerase is loaded onto ssDNA by sliding clamp + Sliding clamp loader
DNA polymerase (function)
Creates new strands of DNA
Adds free nucleotides to 3’ ends of RNA primer in 5’ –> 3’
- Holds onto template and adds nucleotodes to new strands
helps reshape DNA helix by aiding in H-bond formation between bases in template strands and new DNA strand
Can also proofread + correct mistakes that is made
Rate of DNA polymerase
750 BP/second – very fast
DNA polymerase mistakes
Sometimes polymerase makes mistakes –0 adds incoreect nucelotides onto new strand
1 Mistake/10,000 BP
Polymerase = can sometimes fix mistakes –> Halt polymerase activity THEn relax the dsDNA being fromed = expose new templates + new domain of enzyme –> Exonucleose activity = chews up newly synthesized DNA and DNA polymerase restartes adding new nucleotides back on chain (exonuceloase is 3’ - 5’)
After DNA fixes mistakes – 1 mistake in 10^-4 BP
DNA polymerase = very accurate
Fidelity
Acurracey
DNA polymerase = very accurate = high fidelity – have very few mistakes in newly sythesized DNA strands
Leading vs. Lagging strand
Leading Strand = On one template strand DNA replication is in smae direction as the helicase
- DNA synthesis occurs continously
Lagging Strand = DNA synthesis is in opposite direction from reprisome
- DNA replication offcurs until polymerase bumps into RNA primer
- DNA synthesis = discontinous = get okazaki fragments
- Discontinous
Lagging strand end
After replication – Lagging strand has bits of DNA/RNA + breaks in DNA phsphate backbine where 5’-3’ ends of phosphate are not covalently bonded
Closing gaps on Okazaki fragments
END of elongation
Difefrent DNA polymerase (DNA pol. 1) –> recognizes DNA/RNA hybrids – attatches there and cleaves RNA compoennt using exonuclease activity
Polymerase ALSO fills in gaps using DNA nucleotides
Ligase = connects phosphate backbones
Ligase
COnnects phosphate backbone = get continous DNA strand
How longs does Elongation go
Elongation occurs until all DNA on chromsome has been replicated
Where does DNA replication occur
Occurs at each replication fork –> bubble gets larger and larger until entire circulare chromosome has been replicated
Timing in replication
It is impirtant that replication forks end at the same time or else DNA replication will just keep going in circle
Replisome will keep going in circle forever
Issue = replication forks might not finish at the same time = need mechanisms to ensure that replication stops as get haf way around the circle
How to stop replication
Done through Tus/ter sequence on oppositre sides of Ori sequnece
Tus/Ter sequnece
Termination sequnece
Has 2 domains – ter 1 and Ter 2
Tus = binds to ter sequnce
- Tus binds to Ter 1 –> Tus binds to one Ter on top of DNA strand and Another Tus binds to Ter 2 on bottom strands
Tus/Ter complexes = dorectional
Direction of Tus/Ter
Tus/Ter complexes = directional
ONe lines to top of DNA strand and other tp bottom of DNA strand –> Directionality helps guide termination
Example
Ter binds to top of DNA strand –> Replication fork approaches Tus/Ter from either direction (from the top or the bottom)
When Helicase appraches Tus/Ter ocmplex on the same strand that the Tus is bound to (Tus is on top and Helicase comes on the stop strand) = NON-Permissive position –> Helicase is blocksed = whole replisome sits and waits
IF helicase appraches from opposite side (Helicase on top and Tus on bottoms) –> helicase can remove Tus from the complex and continue replicating
At one Tus/ter the helicase is blocked and other noth blocked –> eventually the two replication forms will meet at one Tus/ter complex
- Which depends on which fork gets there first
Euk replication
Similar to Prokaryotic – same ideas just different names
Intiation (Euk)
Euk chromsomes = longer = in order to replicate in suffeincet time = have intiation in multiple positions across linear chromsome
Orgin of replication (Euk)
ARS – Autidory replication sequnece
Euk chromosomes = have multiple ARS
Each ARS = can intiate replication bubble to begin synthesis
In ARS –> recocgnized by ORC (orgin of replication) –> recruits other enzymes to make PreRC (pre replication complex
- Provides location/permission of DNA replication to occur
Goal of initiation in Euk
Goal of intiation in Euk = same as prokaryotes –> open dsDNA to provide template for DNA replication
Elongation (Euk)
Euk = more complicated than bacteria
Have more than 10 DNA polymerase –> Difefrent polymerase for leading/lagging strand
Unwinding DNA in Euk
Unwind dsDNA – more complex because DNA is round around histones to produce nucleosomes
Nucloesomes need to be diseembled and immediatley resembled following replication (Disembled + Reasembles siumlatneous)
Key differences between Prok and Euk
Termination
Termination of linear ends of chromosomes
Telemerse sequence – end of telemerse = has region of ssDNA on 3’
Have 3’overhange – probelm for DNA replication
DNA = 5’ –> 3;’ – only way to replicate DNA in overhang is if habe primer sequnce to the right of 3’ end
- No primer for DNA polymerase = overhand won’t be replicated = with each round of replication chrosmome would get shorter
How do Euk cell replicate the telemere
Telmerase
Telemerase
Ribozyme (enzyme that has protein Subunit and RNA components)
- RNA component = complemnets to overhang = allopw telermase to bind to end of telemere
After binding RNA compoennets still conatins ss domeain
Telemerase = used own RNA compoenent as template for DNA synthesis adding nucelotides to 3’ overahnd = extends ends of chrosmomes
***Telermerase = sepcial kind of DNA polymerase
Binds to overhand and synthesized DNA complememtary to RNA –: extends the length of hthe chromsomes to ensure the overhang can be replicated
Makes telemer sequence longer
If telermases exteneds length of chromsome why fo telemerse get shorter during aging process
Germ line cells express lots of telermase so chrosmomes ends up in gametes have long telemers
Somatic cells = express less telermase
- Without telermase chrosmomes do get shirter each round of replication
- Age = increase rounds of replication = shoter telemers
Increasing teloermase as age defying porcess
Might say that increasing telermase in cells could be age defying process
Issue = some cancer cells avoid cell death by expressing high levels of telermase = telermase might not act as perfect fountain of youth
Use of Telemerase
Allows chromosomes length to be miantained from one generation to the next
Maintain length in species
Structure of DNA + DNA replication
The structure of DNA provided a way to understand how DNA can be copied during inheritance
Structure of DNA
Helix
A-T
G-C
BP facing in
Copying mechanism of DNA
1 peice of DNA –> 2 peices of DNA
DNA replication = semi-conservative replication
Methods proposed for DNA replicatons
- Conservative – Old –> get old + new
- Semi-conservative
- Know this is true
- DNA –> Pulled apart –> DNA synthesis on each strand
- Dispersive
- Mixture of chunkcs of old + new scattered
- No one thought this is what actually happens but posibility
Meselson + Stahl expeirmnent
Idea – First grow bacteria in N15 so that all DNA is heavy –> THEN shift to N14 (light)
- N15 = radiolabeled (heavy)
Looking at what happens to band
Start = All DNA made have N15 –> THEN run on gradietnt gel
1 – Dispersive (would just be a mess)
2 – Semi conservative
3 – Conservative – band for heavy + band for light
Expected results from Stahld Experiment
After two rounds:
If conservative – Always have heavy –> Get more light but no middle band
If semi-conservative –> Still distict bands L/H but start to get more light
Dispersive –> Still smear
Gel in Stahl
After 40 Minutes – ALL middle (no heavy)
As replicate more – newly synthezied used N14 = get light eventually all light + some middle
- Middle never goes away (Always there)
Goal + end product of each Phase
Intiation – Unwind DNA – DNA = supercoiled –> need to get ssDNA to copy it
- require energy
- get ssDNA template
End product = ssDNA = can replicate
Goal = create ssDNA
End = replication forks
Elongation
Goal = synthesize new DNA
End = have newly syntheszed DNA (Nascant DNA)
- Bacteria –> Linear fragment
Goal: Repliacte Template DNA
End: Linear dsDNA
Termination
- Goal = finish replication
- End = get dsDNA end prodyct
Goal : Stop replicating DNA + glue to get circle
End = get circular DNA
List things needed for each step (in order) + State general use of each
Initiation:
1. Gryase –> Unwinds supercoil
2. Energy (ATP) –> Energy to unwind
3. Ori Sew –> Where DNA A binds – Replication starts at Ori Seq
4. DNA A –> binds to DNA A box
5. DNA A box –> Part of Ori Seq
6. DNA B –> Helicase – keep unwindng DNA
7. ss Binding proteins –> Stops dsDNA from reforming
8. DNA C –> helicase loader
9. Template DNA
Elongation:
1. primer
2. DNA pol.
3. Sliding Clamp
4. Template DNA
5. Primers
6. Primase –> creates primer
- pOlymerase can’t start from nothing – need free end to add nuceloties to
- Primase = builds RAN chain from nothing
7. Sliding clamps loader
8. Free nucleotodes
9. Energy (ATP)
10. Ligase
Termination:
1. Tus
2. Ter
3. Energy
4. Ligase
Intiatian
DNA box bind to ori = caises to bend = tension = pops open = get ssDNA
To load helicase on = cut open –> wrao around –> hook toegtehr again (loaded by DNA B loader (DNA C)
Elongation
Have fork –> recurit primase + recurit Pol –> Build DNA chain
Helicase recruits primase THEN DNA polymerase = get nascant DNA
- Held together with slding clamp
H bonds between BP
A-T –> 2 H bonds –> region in ori is AT rich – where dsDNA opens to be ssDNA
G-C –> 3 H bonds
Direction of replication
5’ –> 3’
Leader strand – Towards fork
Lagging strand – Away from fork
- Goes until bumps into primer –> get okazaki fragments
Fidelity of DNA Pol.
DNA pol. = high fodelity
Might make mistakes but can correct BUT somtimes mistakes get through
Termination (bacteria)
Replication forms will meet –> won’t meet at exctley the dame time
If didn’t meet at the same point = would keep going in cirvle = need to end at the same time –> does so using the Tus/Ter
Tus binds to Ter –> have 2 Ter sequenceies (one on top and one on bottom)
- If Tus is on the top strand and helicase is on top = helicase is blocked BUT if helicase comes from the bottom and Tus is on top = hits the Tus off – will be blocked by other tus
END – one will be blocked until other catches up
- End = ligate 2 fragments together
If kept at high heat = wouldn’t need ss Binding proteins
Don’t need ligase if making short thing
Don’t need any of them
What do we need to make short DNA
DNA
Primer
Nucelotides
Energy
DNA replication in test tube (not PCR)
1 single stranded template creates 1 dsDNA product
Heat = get ssDNA
Add primer + DNA polymerase = get new DNA
END = get 1 ssDNA and 1 dsDNA
DNA polymerase falls apart when the temeprature is raised – need a new polymerase to make a second round of synthesis
What do you need for 2 rounds of DNA synthesis in a test tube
DNA polymerase falls apart when the temeprature is raised – need a new polymerase to make a second round of synthesis
Sanger sequncing
Sanger = invented method of sequnecing DNA
Small qunatities of radiolabeled nucleotides that blcoked DNA sythesis were added to synthesis reaction
- Add nuloetides that stop chain seq.
The synthesis mictire would contain difefrent lengths of DNA depending where the ddNTP was incorportaes – these different lengths would be seperated by a gel
By Running difefrent reactions with different ssDTPs the sequence could be “read” dorectlet from the gel
***Need 4 tubes
Issue in sanger seq
Slow + can only sequencey short regions at a time
- Can’t multiplex reactions
- Would need 1 million reactions to sequnce DNA)
Results of Sanger
See gel
Ex. – have end # of Gs OR get band that ends in C
Chain termination by ddNTPs
If have ddNTP = no OH = stop extension
- Add ddNTP = can’t get next nucleootide
Fluorernstley label ddNTPs
Change in sequencing
1977 – radiolabeled ddNTPs + Gels
2023 – Flourecent ddNTPs + capilary tubes/compures
Sanger = the most accuarte methods of DNA seq
Importance of PCR
Considered one of the most important scientific advances in molecular biology
Kary Mullis
Invented PCR –> Was a biochemist working on chemical synthesis of oligios (primers)
Likes surfing + Skiiing + LSD + WOmen
After his BS = he spent a couple of years writting fiction
During grad school he took leave and managed a bakery
Synthesized recreational drugs in spare time
FOUND – that if he used 2 prismers and a DNA polymerase that could wistand high temperatires he could amplify the amount of DNA sunthesized exposnetially
What did they need to make PCR
At time = they got bacteria from hot vent –> proteins don’t fall apart at high temperatures
Kullis = realized that he could have 2 primers + use DNA polymerase taht doesn’t fall apart when hot = could have more than one round of replication
PCR replication
dsDNA –> Heat –> prmer binds –> New DNA is synthesized on top of old DNA –> keep doing cycle
Exponential growth of DNA
Kary mullis was…
Intolerable genius – fought with nearly everyone (bosses +_ labmates _ security guards + receptionists)
He turned his back on scinece
Arugues that AIDS and climate chnage were hoaxes
Importance of PCR
Medicine + Forensices + Agricultiure + Infectious disease + research
Steps of PCR
LOOK AT Pre-class (REAL)
Answer: B (Need inwards)
Primer = 5’ –> 3’
