Lecture #2 (DNA replication) Flashcards
History of DNA discoveries
Start - no one cared about DNA
Avery and MacLeod – Found DNA is the genetic material
- Before thought DNA was a polymer ; reserach only focused on enzymes
Chargaff – Found that the ratio of A:T is the same and the ratio of G:C is the same (Rule of base compoistion)
Hershey Chase – Confirmed DNA is the genetic material
Franklin + Waston + Crick + Wilkins – Determined DNA structure (DNA is a double helix with anti-paralel strands)
- Provided a hypothesis for how DNA could work as a template for replication
Meselson Stahl – Foun that DNA replication is semi-conservtaive (each doughter stand has one parental strand and one new strand)
Kornberg Ochoa – Isolated DNA polymerases (Set the stage for mechanistic understanding for how replication works as a biochemical process)
- Gave rise to findings that all life comes from inanimate matter
Replicon model
Idea for how replication gets started (idea for how intiation occurs)
Brenner and Cuzin model (replicon model) - Have some peice of DNA – on that DNA have a plave where replication will begin (Now known as the ORI site) –> intiator binds catylzye the work of opening parental chromsome (seoerate dsDNA into ssDNA) –> get bidrectional forks (two forks mving away from each otehr) –> replication proceeds to expand teh bubble
- Ori site – Cis acting element that recruits a trans acting protein (recruits intiator)
- bubble = theta structire if in circlar DNA
Ori site
DNA seqeunce that stays strat to assmeble replisome here
Cis acting element that recruits a trans acting protein (recruits intiator)
Do you only need 1 orgin to replicate
E. Coli – gene is 4.6 Mb ; fast fork speef (60 kb/min) –> expect that replication will take 40 minutes
Yeast – genome is 14 Mbp (bigger than E.coli) ; fork speed is 3 kb/min (slower than E.coli) ; if had 1 ORI it would take 80 hours to divide (we know it actually takes 20 minites)
Humans – If had 1 ORI it would taje 1 year to replicate
Overall - Overall – real replication times are must fatster than expected because organisms with bigger genomes have more ORIs (Euk and Archea have multiple ORI sites)
Replication Forks in Euk
Forks in Euk are slower because they ahve to contend with chromatin
Southern Blot
Overall - shows DNA in sample corresponds to a sequence of interest
Run Gel –> Denature –> Add DNA with known sequence that has a radio iostope attached (Probe) –> hybrize the probe with DNA in gel –> See if have band (Shows what sequence is found and what they look like in cells)
Mapping replication Orgin Experiment
Finds replication orgin in the region of interest
Cells –> Shear DNA –> Run southern in 2D (gives size and structure information)
- 1 direction – run top to bottom
- 2 direction – trun the gel and run DNA in the other directiion
Can run native DNA in 1st direction and then denatire for the secon direction
Can predict the kinds of structures found in popultion of cells undergoing replication
Types of replication origins + Results form 2D gel
- Forking coming in one dorection
- Buble (replication starts in middle of DNA)
- Forks converging
- Offset bubble
ALL have diferent DNA structures (Y shapr or bubble shape) –> Each sturcture gives a specifc second dimersion
Chart :
Get arcs and discontinuities – tell you if you have a fork or bubble etc. For your region of interst
- Can distuguish the sturcture by 2D gel – Ex. See 2 forks converging
Reseracers mapped eveyr replication orgin in yeast
Each structires gives rise to a fingerprint of mobility
Only looks at one or two regions at a time
Reserachers did this to map every yeast ORI
Replication Bubble during replication
Replication bubble – expanding as you replicate DNA –> path cells at different stages (different cells have bubbles in different regions or differetnt sizes)
New Method for mapping orgin of replication
Sequence to find orgin (used for metazonas organisms more rapdily)
Give cells pulses of EdU to put EdU into replication forks – use EdU labeling and selectivley pul put okazaki fragments
- EdU = Uracil analong (with ethano group) that is recognzied by Antibodies or can be used to do chemistry
Denature and fractionate DNA –>Use Biotin to Pull on EdU get rid of RNA THEN see what DNA is being synethsized and where it is being syntehsized
End - Sequece to map eatly/late intiation and termination zones (Find zones of intiation + Eloingation + termination)
New Method for mapping orgin of replication (Results)
B - Data –> Starts with over represenattion of one strand then transition to overreprentation of the otehr strand
- Patterns shows if intiation or if temrinating
Patterns:
- Low to high –> intiation
- High to low –> termination
- between is elongation
Chart at bottom shows how to read the data
What did we learn from mapping orgin of replication
Human intiation replication stochastically in broad non-transcribed zones
There is no sequence basis for itiation site in humans
-Intiates based on context and chrmoatin status
How to start replication (Intiation factors)
ALL Enzymes that will start syntehsis
Intiator – bind to origins (ORI) and recruits heicase
- Bacteria – DNA A
- Euk – ORC (Six SU complex)
Helicase 0 unwind DNA
- Bacteria – DNA B (6 SU complex)
- Euk – CMG (11 SU complx ; MCM2,Cdc45,GINS)
Helicase Loader – Loads helicase
- Bacteria – DNAC (with DNAA)
- Euk – Cdc6, Cdt1 (With ORC)
Primase – Makes primers (DNA dependent RNA Polymerase)
- Bacteria – DNA G
- Euk – Polymerase alpha (Pri1, Pri2, PolA1, PolA2)
Why does DNA replication need primase
DNA polymerase can’t do the 1st nucleotide condensation step to intiate suntehsis = needs primer (needs 3’OH that is already annealed onto template)
RNA Polymerase can carry out the 1st condesation step
- Primase is an RNA polymerase
What do all Intiator proteins have in common
ALL use NTPs as a substarte (As synthestic substarte or to power aspct of catalytic reaction)
- Means they are all motor proteins or molecular switch and use NTP to control that
- Primase syntehsize product with NTP
Most of AAA ATPases – AAA ATPases conrtol intiation
ATPases Assoaited with various Cellular Activities (AAA ATPases)
ALL have Common Nucleotide binding fold (extra alpha helical)
Looks like a clam shell (nucleotide sits in Jaws of clam shell)
- ALL HAVE extra alpha helical bit distinguishes AAA family (structure is capped with helix)
Work in many different procces (Ex. Nerve firing + protein degradation + DNA replciation and transcription + chromatin remodling)
ALL AAA ATPases enzymes serve as macromeular motors (move along substarte) or remodling factors (chnage structure of target that they act on)
What do all AAA ATPases form
ALL form higher order rings shaped oligiomers
Usually Closed hexameric rings (6 SU which DNA sitting in middle)
- Example – enclose DNA
Can also form cracked pentameric rings (open ring)
- Ex. Clamp loader
FOR BOTH - Oligomeric Rings encircle the substrate that will be acted upon)
Why form oligiomers - Forms oligiomers because active side of ATPase SU can bind ATP BUt can’t hydrolyze it so it needs help from partner SU
AAA ATPases active site
AAA ATPases generate a Bipartite active site
Bipartite active site – means that the nucleotide binding fold of one SU (where ATP binds) has many elements that binds nucleotide and coordinates hydrolysis
Active site has consserved motifs with specifc functions:
1. Walker A motf – Binds nucleotdes
2. Walker B motif – Hydrolyzes nulceotode
3. Sensor 1 – Hydrolysis
4. Sensor 2 – binding/hydrolysis/coupling
ALSO have a second SU in AAA ATPases
Second SU in AAA ATPases
Overall - donates an extra Amino Acid
Second SU = Arg-finger – used for hyrolysis/coupling
Angenine finger – Amino acid is donated to partner bipartide active site to sense if ATP is bound and to assist wth hydrolysis and sense when the phosphate is release and you have ADP (allows the second SU to know what the bipartide SU hydrolysis staus is) –> Causes signals transmited through the ring to cause conformation chnages
- Arginine finger can also pater with Bipartide SU to move with respect to each other –> then the whole stcuture will move because during ATP binding and hydrolysis = get confirmation change –> -> confirmation chnage can be used to do work
Replicate Helices Structure
Hexamer Ring
Bacteria - DNA B = Homohexamer
Euk – CMG = heterodundecamer (11 SU)
- MOtor portion of CMG = hterrohexamer ring (MCM227 complex)
Helicase = AAA ATPase = Motor part = leads the replisome foward
Issue - How do you get these onto DNA (If DNA is long = cant thread onto one end ; if you are a cirle fo DNA then can’t add –> something need tso open)
Intiators/Helicase loaders structure
Intiators/Helicase loaders are sprial/ cracked-Ring ATPases
Open helicase with helicase loader
Intiatoer + helicase loader – have cracks (NOT fully closed)
Cracks allow ATPase ring to encricle DNA and keep helicase rings open –> THEN crack the Helicase ring open to put helicase onto the DNA or can close an already open helicase ring at the right spot on the chromosome
Helicase Loading - Bacteria
Have DNA A box – Origin have sequence specific DNA binding elements for intiatory
- DNA A box binds DNA A
- Flanked by AT rich region (more prone to opening)
- DNA A initiator binds to region –> wraps region to helical structure –> Opens the AT rich region (extnds sprial oligimer extrends into the singel starnd regions to keep bubble open)
Bubble creates binding site for DNA B (helicase) and DNA C (helicase C loader) –> forms complex that comes in and interface with DNA A/Orgin complex
Can get Helicase loaded
ALL require ATP - DNA A , DNA C = require ATP
Helicase loading - Euk
Orgin recoznition by intiator (ORC) -> ORC is joined by a CDC6 (closes orc ring) –> AAA ATPase SU forms cracked ring that binds DNA –> CDC6 seals this to create a link between ORc, CDC6, and DNA (serve as landing pad for MCM helicase) ; MCM helicase is already cracked open (Chaperoned by CTC1 that keeps it from interacting with the wrong elements of DNA)
ORC and CDC6 and CDT1 chaperone (put) two copies of helicase onto DNA BUT do this onto dsDNA
- MCM form double hexamer (have 2 6 SU coplexes of MCM rings touching each other) - can slide around and ecnrile around dsDNA BUT don’t fall off because encricle it
- Hexamer persists until S phase
Once MCM double hexamer is in place it can be activated –> Accesory subunits will come in (kinases and chaprones will trun things on)
- At S phase SU will come in and bind to MCM
END – have 2 CMG complexes that seperate form one anotehr and move away from one anotehr as the fork begins to mature
- At the same time the helicases are NOW on ssDNA (kicks out one strand and only surrounds 1 strand)
- dsDNA –> ssDNA transition is unknown
Why do Euk complete Helicase loading differentley
Done to seperate the loading phase and the activation phase of helicase
Loading = G1
Activation = done in S (activate MCM)
Do this so that they can’t over replicate the genome (prevents rereplicating)
- Because it takes a long time to replicate DNA - IF intaitaior came to start replication before the first round is done then you would have over replication of some segments of genes = get CNVs = get chromosomal instability
Loading Overall - Bacteria
Overall – DNA C opens DNA B ring to allow DNA to enter (DNA B –> DNABC –> DNABC on ssDNA)
DNA B (helicase) Starts as a closed ring –> DNA C (Helicase loader) binds to DNA B –> Binding of DNA C causes a confirmation chnage that opens DNA B (helicase ring) –> DNA is able to go throught the center of the ring (ssDNA binds to the whole system)
When DNA binds to helicase –> Causes a cofirmation chnage in helicase –> causes confrimation chnage reisomerizes to topologically close back the ring and link to the ssDNA (causes DNA B (helicase) to drop down –> Seals helicase ring on DNA )
- Other things come on board to kick loader off (process is coupled to ATPases)
Loading Overall - EUk
ORC forms confirmation states (inactive –> oslates to active configration where there is a whole that oepns that can bind DNA (DNA IS bent) ) –> hole binds DNA then CDC6 to encircle it and close hole –> can get MCM with CDT1 that are prepared – MCM/CTC1 will dock at angle (angle allows the MCM ring open and slot onto DNA that comes out of teh center of ORC chnael)
- AS MCM comes on you have intermediate that called RCCM complex – stage where ATP hydorisis is KEY
ORC flips over to the other side and does the same thing on the otehr side with a second MCM to form double hexamer
- ORC jumps over MCM and repeats the loading process on the second side
What prevents Orgin from Refiring in Euk
Have regulation at intiation to make sure replication refiring doesn’t happen
Regulation mechansims :
1. Post trnslational modifucations (common in yeast and humans to turn factors on/off)
2. Proteolysis (degrade SU at certain points to make sure they is not enough to cause refiring)
3. Nuclear export
Regulation has different proteins in difefrent organisms –> MEANS that the mechansim of intiation is consrved but regulation evoloved to be different (regulation is not conserved)
Slide - shows yeast vs. Metazoans (many things happening)
Bacterial regulation of intiation
Control ONLY at the level of DNA A:
DNA A – needs to be in ATP state = bind sto ORI and promote self assmebly
- Formatino of ATP state sets it up to hydrolyze nucleotide to get ADP form
- DNA A = product inhibited enzyme –> when hydrolyzes ADP get stuck = destabilizes assmeby and prevents reintition
- DNA A need sto be reaspened – done by cardiolipid
Cycle of ATP –> ADP
- DNA A is active with ATP to bind ORIc
- DNA A is switched off or reactivated by exogenous factors (have proteins that affect if DNA A will hydrolyze ATP)
- Affectors in cells regulate ADP xchnage
Bacterial regulation of intiation at the orgin of replication
Orgin itself is subject to control that prevents rereplication
Portein that binds to methylated DNA
- Syntehsized DNA = not methylated – need to add methyl marks later
- SeqA Protein binds to methyalted orgin region and prevents DNA A binding
Replication elingation Proteins
Polymerase = works with accesory factor that anchors polymerase to DNA – clamp
- Polymerase synthesize daughter strand from parental template
Helicase = at the front end of the fork
Clamp = ring –> needs to be loaded
Replication elingation Proteins Evolution
Enzymes that catylyze strand synthesis are not homologous between bateria and euk
- Would think replication would be conserved BUT when ancestor split to archea and bacteria - one bacteria got one set of replication proteins and archea got a different type of replication proteins
- Don’t know which line is THE ancestral
- Helicase + polymerase + primaser = diferent evolutionary linease in bacteria vs. Eukaryotes
Clamp + loader + ssbp + helicase loaders = conserved
Replciation evolvled twice (different from transcriotion and tranlsation which are conserved)
Function of Helicies
Replicative helicies drive strand seperate
- Helicase = encircles and moves along single DNA strands
Bacteria and Euk Helicase translocate on opposite strands in opposite directions
DNA B and CMG move on opposte strands in oppposute directions
-DNA B goes in 5’-3’ (lagging strand)
- CMG goes 3’-5 (leading strand)
Helicase is a rotary motor
Helicases = use conversed rotary engine mechanism
6 Su will each turn in ATPase cycle to propel helicase on the template
Image – shows the motor SU (pink is nucletide)
Have a varierty of ATP binding states in one ring (Some active sites have ATPase ; some are bound to product (ADP) ; some are empty) –> acting by a rotaerty hydrolysis
- Rotery hydrolysis = have site thas empty then bind ATP then hydrolyze ATP and release product ; as that SU is working anotehr SU is ALSO hydrolyzing –> goes around
- Wave of ATPase activity that goes around
Nucletide binds and exchnage in SU – puts active site through ATp rasnition and product state
Nucletide binds and exhnage in SU - puts active site through active ste through ATP transtion and product state - as theyr go through the SU flex = allows teh motor SU to craw along teh substarte they are bound to
= hae motive force to move enzymes along linear track
DNA polymerase families
There are many DNA polymerase families
Two major families:
1. A, B, D, Y, and RT are related (for one sueprfamily)
2. C and X are related (form a superfamily)
C is replcative in bacteria but X is repair (famil C is in bacteria)
B is replacative in Euk and Y is involoved in repair (B is in Euk)
Have a flip flop with what is replcative and which is repair
DNA polymerase structure
Polymerases = all have a common structure
Overall - looks like a right hand
- Palm = catalitic fold – where the action for synthesis is (where substrate sits)
- Thumb – Rigid brace (immobile)
- Fingers (middle domains) - flexible (mobile)
Allows polymerase to grip DNA and insert dNTPs as carries out polymerization
What does DNA polymerase use to function
DNA pilymerase = works by encorporating nucleotidea through metal depent process (uses three divalent ions – includes Magnesium)
Three metal mechanism arrow pushing
3rd metal ion helps promote the second metal ion to leave
Once the second metal ion leaves it disfavors bond reformation
- Helps push the reaction foward (otherwise DNA would go back to equilibroum)
How do polymerases distiguish teh correct bases (experiment)
Idea - Polymerase can sense the strength of the H-bond to know if the base is right (H-bond dictates the fibeility of DNA polymerase)
Tested idea using isosteres of normal base
- Isosteres replace H-Bond donor acceptor groups with a group the same size/shape but can’t make the same interactions (no H-Bond)
Do experiment where you see if teh DNA polymerase are still as acurate with isostreres as they with normal bases when syntehsize DNA
- Use radiolabled nucletdes to see if the correct base is added in the template
Image - uses a Thymine anology
- When template has an A –> get T added
- When template has a C –> get a G added
- When template has isothere T –> get A added (same as normal)
Quntofy - see that polymerase are not recognizing H-bond they recognize shape (sense sterics)
- Very energtically unfavorable when have the wrong shape
How are Polymerases held in place
Polymerases are held in place by sliding clamps that slide along DNA iwth polymerase
Clamps = ring shaped proteins that encicle the newly snthesized DNA as it exits teh polymerase active site and tethers the polymerase to imporve processivity
IF Polymerase accidentley falls it is still tetehred to DNA because ut us tethered to the clamp and the clamp won’t fall off because it encircles the DNA = Polymerases can quickly rebind if it kinda falls off
How are the clamps loaded
CLAMPS ARE LOADED BY DEDICATED CLAMP LOADERS
Clamp rings need to be put on by the AAA ATPase deoendet protein (Clamp is put in by the clamp loader)
Clamp loader use ATP turnover to control clamp opening/release
- Clamp loader open in nucletide free state –> binds ATP puts in confirmation where it can bind t clamp and open clamp –> looks for primer template junction –> because notch shape it can slide on to primer template junction –> bound to junction it causes te ATPase to hydrolize ATP –> causes clamp to dissoate and close ring
Leading vs. lagging strand image
Shows helicase unwinding DNA ; Polymerase is moving down
- One polymerase is stuck going in the wrong direction (going away from helicase) = DNA has to be syntehsized in segments
- See leading strand coming in towards the helicase ; lagging strand the DNA template is drough in in the worng orientation
- Lagging strand synthezued okazaki fragments – Discontinous segments need to be primed individually by primase –> primase will make RNA and then DNA polymerase will come in and make the fragment
- Form short okazaki fragments (vary in length depending on organism)
Leading and lagging strand polymerases
Leading and lagging strand polymerases are linked
Bacteria = Linked through the clamp loader
- Clamp loader has long linked elements coming off ATPase SU –> Grab onto DNA polymerases + Helicases = holds the fork togetehr
Euk - CTF4 or AND1 protein – links leading and lagging strand polymerases together
- Leading strand polymerase is coupled to helicase (CMG) through SU/SU interactions (no intermediate adapter)
Issue - WHat if one of the polymerases run into a probelm (what does the other do)
Testing to see if the DNA polymerases are coupled/communicate
Use single molecule analysis ; form rolling circle replication (used to study fork dynamics)
Rolling circle:
Take plasmid –> tetehr one end of the plasmid to cover slip (plasmid has gap so can add replication fork with helicase/clamp loader/polymerase in the gap)
Helicase will encircle the DNA and move foward and unwind the strand that has the knick
DNA polymerase grabs onto the 3’ end of the knicked strand and extends it
If the leading strand gets made –> causes the template to elongate ; lagging strand polymerase comes = get short lagging strands made on top of the leading strand (forms intact dsDNA)
End - can have simultaneous single molecule analysis of leading and lagging strand synthesis
Replication fork (Image)
Replication challenge (Chromatin)
Issue = chromatin
There are histones that need to be removed from the fork BUT the histones have epigentic infomration on them
The epigenetic infomration needs to get onto the new daughter chromosomes in a way that preserves the epigenetic infomration
- Old and new histones are equally apportioned between daughter nucleosomes –> Preserves epigenetic information between cell divisions
How is Replication challenge (Chromatin) dealt with
Replisomes have many things appended to them including a domain in DNA pilymerase Epsilon that allows the Polymerase epsilonto get parental nucleosomes and deposit onto the leading strand
DNA ply episolon is in competition with MCM2 and CMG helocase that is ALSO grabbing the histones and putting them on the lagging strand
- IF mutate the MCM arm then all histone go to the leading strand
- IF mutate Polymerase epison the histones all go the lagging
End – get 50:50 distubution of parenta nucleosomes on the two daughter strand + Remainder are filled in with new histones by CAF1 (histone chaparones)
Replication challenge (Copying chromosome ends)
Replisome can’t complete synethsis at ends of linear chromsomes (at telemeres) –> Solve problem with telamerase
Problem:
Leading strand fork goes to the end by helicase with no issue
Lagging strand can primer up to the end BUT the RNA primer has to be degarded leaving a gap –> gap neds to be filled or there will kep getting a shorter arm at the telemeers –> telemers would get too short = bad (get NHEJ and chromosomal instability)
Solving Replication challenge (Copying chromosome ends)
Uses telamoerase (RNA dependent DNA polymerase)
Has own template (Telmerase RNA = template)
Telamerase binds to and end where there is a single strand gap and have repetative telemeric sequences
RNA pairs with portion of end that allwos polymerase portion to extend it t the end of the RNA –> enzyme hops through translocation to the end carrying some RNA at the end of teh DNA that was just syntehsized and extends teh DNA further and resytnthesizes the whole set
Does local replicatiinon and repeat addition = Adds repeats to teleermes through hopping mechanism = telemeres have repative DNA
Replication challenge (Okazaki Fragments)
Overall - Need to remove RNA and heal DNA
Some polymerases (Pol 1) have 5’-3’ exnuclases = chew up RNA and then fall off laving a nick –> Nick is sealed by ligaes
Some polymerase stop when they hit RNA and recruit RNAse H = leave a gap
Some Polymerases can plow through RNA/DNA suplex and leave a flap strcuture –> flap endonucleases can come in and clean up –> ligate nicks
***Mutations in enzymes gives rise to diseases (premature cancer or aging canse ties to DNA repair)
Replication challenge (Converging forks)
Issue – how does the fork know where to end
Solution (NOT only one happens):
1. Have proteins on DNA = forms a road block
2. Forks collide and bypas each other –> they see that they passed another fork and stops
3. Superhelical tension builds up in DNA = inteferes with fork progression
Replication challenge (DNA topology)
Because DNA is a double helix any machinery acting on it has to deal with coiling and tangling that DNA is prone to
- Causes torsional strain
As the replisome is unwinding the DNA the DNA will form positive supercoils in front and negative super coils behind
ALSO have two strands of DNA being synthesized behind the replisome - the replisome can rotate with teh DNA –> as replisome rotates the synthesized DNA can get tangled (Tangling = Precatination)
- Newly synthesized daughter strands can tangle with one another
Affect of Twist and Link
Differences between twist and link leads to 3D coiling of the double helix about itself = creates Writhe
Twist + Writhe = Link
If you chnage twist BUT keep link the same then Writhe must hange or visa versa
If chnage link – Twist and writhe can both chnage BUT for B form DNA Twist always stays 10.5 so WRITHE always has to chnage = 3D coiling of the
Closing linear DNA into cirecle
Take linear DNA –> ligate the ends –> if the numver of truns is enough to get relaxed state = get closed relaxed circle (Writhe = 0 – no circling of the dsDNA around itself)
IF you take the DNA ends and you delibertaley twist them (Ex. remove 2 truns of DNA ; Lk = -2) –> join the end –> Get bubble (Tw = -2) –> DNA does NOT like this (wants the bases to refrom) –> NOW chnaged the link number by 2 and the twist number by 2 BUT this won’t stay Twist will become Writh (Wr = -2) –> get negtaivley supercoiled DNA
- Turn the -2 twist into -2 writh
IF overwind and ligate together –> get positive supercoilnd DNA
Affect of Supercoiling on Replicated DNA
Replicated DNA must be unlinked (chromosomes can’t have links)
When two forks onverge = builds positive supercoils in front of them (forms interwtings) –> DNA coils around them –> something needs to come in and unink them otherwise can’t seperate chromomes (DNA would break)
Numer of links = #BP in the system/picth
- Number of links is propertional to number of turns in DNA (every turn is 1 link that eeds to be removed)
6 GB of DNA = >500,000,000 links must be removed from DNA (from replicated chromosomes)
- 1 link left over in mitosis can cause the chromosome to break = kill cells
- In linear chromsome some links will just fal apart but circular chromome every link needs t be removed
How do convergig forks deal with unlinking (Experiment)
Xenopus egg extract DNA replication system –> Can add/remove specific factors —> Can remove DNA at different times, monitor replication status down to single base resolution
Add plasmid –> start replication –> ADD a block (lac repressor binds to DNA sequence unless you give lactose and the repressor comes off) –> fork stops at the at the repressor but then remove yhe repressor –> see what the intermediate DNA loosk like –> resolve DNA states on gel
- Depends on if add RE or not = can distiguish betwene which regions are replicated or not
Can change factors and see what is needed for teh forks to move past each other
Get single basde resolution by sequencing to see what replisome is doing
END - found that Eukryotci forks approach each other and then move past each other
Replication termination
Two CMGG assoiated forks apprach -> DNA in middle becoems unwound by Topisomerases –> forks move past one another –> at bypass point the helicase hits a gap and then dsDNA (runs into okazaki fragment) –> helicase can run over the okazaki fragment = get two strands of DNA in teh center of the helicase –> RNA is remved and patch DNA with ligase –> Helicase has lost ssDNA that it displacing and is encirling two stands = gets biqunated and then degraed
- The absence of a displaced strand leads to MCM ubiquitination/CMG dissolution
Eukryotic cells = use replisome bypass system to control when replisome stops
Have DNA BUT still have the supercoils in front of the replisomes and catenanes that need to be resovled (need to untangle links)
How do you get rid of the supercoils
Uses topisomerases
- Can also add or remove knots and catenass from DNA
Two Types:
Type 1 = cuts 1 string of DNA
Type 2 = cuts both strings of DNA
BOTH form covalent enzyme (tyrosyl)-DNA intermediates (uses active site tyrone)
- Make sure they stay tethered to DNA to prevent them from dissocoated once it cleaves DNA BUT they can also get stuck which causes a protein DNA leison (doesn’t release the broken ends)
Detecting Topoisomerase Activity
Uses Native Gel electropheresis - partitions based on DNA shape
- Seprate based on shape and size
- Can use to monitor (de)catenation and (un)knotting activities as well (monotor topoligic states) + monitor supercoils
Run gells - seperate topological state of DNA –> see knotted or supercoiled
Image -
See supercoiling DNA that is progressivley ralexed ; End have 1/2 supercoils in there
- See intermediate species of topisoemrase (Might have a writhe or 1 then 2 etc.)
Topological Conventions
PLectonemic (default state) - thread where you twist and then coils on self
- Negative supercoil = Right have cirality
- Positive supercoil = Left hands chirality
Solenoidal/tooidial (Requires proteins ; Stabilized by Nucleosomes)
- Negative supercoil = Left have cirality
- Positive supercoil = Right hands chirality
Toposimerase Type 1B function
Type 1B enzymes = nick and swivel enzymes (swivilezases)
Type 1 B = C shape – Bind to DNA and clamps around dsDNA –> nick 1 strand using tyrosine –> part of clamp relaxes while the other part keeps grabing DNA –> second duplex (DNA) rotates around the attacked phosphdiester bonds –> eventually stops and gets resealed
- Work by free rotation mechanism –> nick and allow torsion energy to diffuse out through rotation around the remaining phosphodiester binds –> then reseal
Used to remove positive and negative supercoils
Why is ribonucletide incorporation an issue
Topisimerases = reason that incorpation of rNTPS is an isue because topisomerase cut DNA at the 3’ end (tryosone because linked to the 3’ end) - IF you have an RNA in that spot THEN 2’ OH can attck teh phosptyrone intermediate –> forms a cylcic aduct –> can’t be religated
IF TOP 1 hits an RNA –> get DNA damage that need to be rapired –> sometime sin repair process can get slippage that leads to 2 BP deletions
Topoisimerase Type 2 Structure
Constrcted like Jaws (have diferent parts of the enzyme that can open and close)
Have ATP binidng protein (require ATP for function unlike Type 1)
Have DNA binding and cleave segment
Have many structures with mant intermedate confrimation space
Topoisimerase Type 2 Function
Bind to a DNA dsDNA (called G segment) – when sitting on the first dsDNA they can bind a second dsDNA –> ATP binds –> can transport second dsDNA (T segment) through the first dsDNA by cleaving the G segment 1st DNA) –> Get dsDNA break –> open a gap (30 A in first DNA) –> pass second DNA dsDNA (T segment) through gap –> Seal broken DNA and expel the peice DNA throgh exit chnael in enzyme –> ATP hydrolyzes and system resets
Can remove positive and negative supercoils + can unlink DNA tangles (remove Catenanes) –> make sure chromsomes can be pripery segregated
