Lecture #2 (DNA replication) Flashcards

Question

Loading Overall - Bacteria

Answer 1

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)

Answer 2

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

Answer 3

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)

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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)

Answer 8

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)

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

DNA pilymerase = works by encorporating nucleotidea through metal depent process (uses three divalent ions – includes Magnesium)

Answer 13

Image – Nucleotide and template is in cyan; Parent chain is in red ; New DNA is green ; Finers are orange Have acidic amino acids and metal ion comes in Fingers moving is coupled to base pair snesing and primer template translocation - Fingers move to get Basepair – fingers lock and get pushed out - Tyrosine pushes the newly added base out of the active site Nucletide comes in – finegrs rock nucleotide to position so can base pair and form bond – as pyrophosphate leaves from nucletide addition teh fingers domain rock back out and the tyosine pushes the template foward by one

Answer 14

Function - Help bring in new nucletide with cluster of acidc Amino acids 1. metal ion hydrolizes and attaches the alpha phosphate 2. ion heklps with the pyrophaspahte levaing group 3. Transient third metal ion (on the oppoiste side of the otehr two ; on top of the first two) - 3rd ion appears before the breakage of the bond between the alpha beta phophate

Answer 15

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)

Answer 16

DNA has two anti paraellel strands Bases interact through H bonds - It was thought that the H bonds would help distiguish correct base pairing (because 2 H-bnds for AT and 3 H-bond for GC)

Answer 17

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

Answer 18

Have active site that can recignize the correct shape of the Base pair (shape of BP is based on the H-Bonds ining up) Image - Correct shape is grep ; mispair is magenta --> see that the mispairs takes on different shapes = acitive site gets mis-shapen --> flexing causes it to lose its grip --> ejects the wrong base - Fingers and thumbs misalogn = don't get good fit By this sensing mode the Polymerase makes very few mistakes

Answer 19

DNA polymerases have exonuclase domains --> when the wrong base enters the polymerase the plymerase can sense it in the active site and knows it is knot the right fit --> Polymerase slows down and the exonucleus wil come in If wring base is already encorparted = cause the pilymerase to interact less strongly with primer template complex = gives time for commplex to leave polymerase active site and go to exonuclease active site

Answer 20

Steric matching ➜ Basemismatch error rate: ~1 in 10^6 Proofreading exonuclease further decreases error rate by ~10^2

Answer 21

Steric can exlcude rNTPs from the active siite BUT the concetration of rNTPs is more abundent than dNTPs (RNTPs = milimolar vs. dNTPs = micromolar) If an rNTP goes into the active site polymerase needs to eject it BUT at some frequencies the rNTPs get encooroated into DNA = causes an issue - Polymerase misenropoarte more RNA --> RNA needs to be removed

Answer 22

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

Answer 23

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

Answer 24

Confirmation chnages of clamp going from ATP bound to product bound (ATP hydrilyzed state) - Clamp become closed when it goes between those two states

Answer 25

Polymerase polarity is a problem for semi-conservative synthesis Replication ONLY go 5’-3' BUT offcurs on BOTH antiparallel DNA strands - One stand has 5-3 polarity ; one strands has 3-5 polarity - One strand has the right orientation (going towards helicase) ; one is in the wrong orientation ISSUE = all polymerases synthesize 5-3' direction Means only one strand is syntehsized continously (leading strand) One strand is synthezied discontinouly (lagging strand) - Primase is needed to syntehsize the RNA for intiating lagging strand synthesis

Answer 26

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)

Answer 27

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)

Answer 28

How is one polymerase able to maintain speed with one another (Does this tether affect the rate that the two move) For a while people assumed that this was true because the leading strand doesn’t finish before the lagging strand = people assumed it was coupled

Answer 29

Might be important for leading and lagging strand synthesis to stay constant because if there is a lesion on the leading strand causing the leading strand polymerase to stall BUT the helicase contines to unwind then the lagging strand would continue making DNA = would result in a lot of ssDNA = bad because ssDNA is more recative Need the polymerase to be coupled in addition to the physical tethering that they already have

Answer 30

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

Answer 31

Each dot = replisome Look in real time at replication fork movement (newly syntehsized DNA gets traced by flow as the replisome proceeds) - Can change condistions (Exl chnage nucletide concetration or withhold some of the compnents) - Can distguish RNA and DNA and see which is leading or lagging strand) Ran expeimrent with different concetrations of primase - If primase is limiting then lagging strand sythesis shoudl slow down --> if lagging strand synthesis should slow down if it is coupled Results – Leading strand synthesis DOES NOT slow down = end with lots of ssDNA - Means lagging strand synthesis with primase is uncouped form leading strand elongation ; reaosn the two strand get made contemperasley with ine another because the leaidng strand stuteres (pauses) --> the discintinousities in leaidng strand syntehsis that it allows the lagging strand synthesis to keep up - Leading and the lagging strand wynthesis are physically coupled but NOT enymatically coupled

Answer 32

Priming (lagging strand synthesis) is uncoupled from leading strand elongation Random pausing/dissociation events maintain polymerase synchrony

Answer 33

Image – shows simplified replisome See helicase encricling DNA and extruding the other (Helicase seperates by displacing one stand physically Clamp loader holds thing two Polymerases together + clamp loader attaches to the clamp - Clamp is attached around the primer being made - Primers make primers – puts to clamp loader --> Primer gets handed off the the lagging strand polymerase which synetshizes the lagging strand - Has dsDNA coming out of polymerase

Answer 34

*DNA ~20Å diameter *Replisome ~3-400Å (polymerizing complementary base pairs) *Replication fork speed ~1000bp/s *Time to complete genome ~40 min (4MB) * Error rate <1 per 106 bases

Answer 35

It is as if there is a fedex truck dirving along path ; you need to sit on back and lay down complenetary colro of stripes ; would need to have 1 botch strope per 170 km *DNA ~ 1m (bicycle path with a striped line containing four colors) *Replisome ~ FedEx Truck (scanning, matching & painting complementary stripes) *Fork speed ~ 600 kph (375mph) *Error rate ~ 1 botched stripe pair per 170 km (106 miles) DNA polymerase has crazy high fidelity

Answer 36

1 L yeast culture ≅ ~4x1010 cells ➔ 400,000 km DNA (Earth-Moon distance) - All DNA in cells end to end goes from earth to moon All the DNA in your body, laid end-to-end, would reach from the sun to Neptune and back…twice A human synthesizes ~2x1013 km of DNA (2 light-years) during his/her lifetime All this must be accomplished in a manner that minimizes errors! - Not error free because error free = no evolution = need some error rate but not too much

Answer 37

1. Chromatin 2. Coping chromosome ends 3. Fixing Okazaki Fragments 4. Converging Forks

Answer 38

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

Answer 39

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)

Answer 40

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)

Answer 41

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

Answer 42

A REVERSE TRANSCRIPTASE – ADDS REPETITIVE DNA SEQUENCES (TELOMERES) TO CHROMOSOME ENDS

Answer 43

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)

Answer 44

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

Answer 45

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

Answer 46

Tangling - take cord and wrap it up --> put in a box -- shake the box --> cord gets tangled up - Long Polymers get tangled Coiling - Every time a motor protein unwinds a turn of DNA the DNA will respond by coiling

Answer 47

B form DNA pitch is 10.5 bp/turn = Twist/Tw of DNA - Twist = number of times the two strands twist around each other (1 full twist is 10.5 base) (amount of BP to get to same place but above one verticle level) - Twist does not have to be integral Linear DNA is open bounded BUT can DNA in a cicle (bounded) --> Circle case an issue because you need the 5’ and 3’ ends to aling in order to ligate = need to wrap around each other an integral bumer of times Pitch is NOT integral (can be decimal) BUT in a closed circle the two strands can only wrap around each other an integral number of times Linking number (Lk) = number of times strands wrap around each other (always an integer when DNA is closed ; can’t be a decimal)

Answer 48

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

Answer 49

Plasmid = has supercoiling --> if you cut the plasmid to be linear then the supercoiling is lost because the ends are free (plasmid linking number can chnage BUT it is mostly due to writhe)

Answer 50

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

Answer 51

Longer DNA can have a writeh f 2 and it won't be shortned BUT shorter DNA with a writhe of 2 with be compacted

Answer 52

RNA pol makes positive supercoils in front of it but because it is not making new strands it makes negative super coils behind it

Answer 53

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

Answer 54

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

Answer 55

Xenpous egg system = has all the things needs to carry out replication --> can add or remve factors and see how rpelication intiates or elongates or temrinatison or DNA repair

Answer 56

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)

Answer 57

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)

Answer 58

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.)

Answer 59

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

Answer 60

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

Answer 61

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

Answer 62

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

Answer 63

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

Answer 64

Crystlize the proteins and do studies