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DNA replication
Eukaryotic Prokaryotic
DNA replication
DNA replication
• DNA replication, the basis for biological inheritance, is a fundamental process occurring inall living organisms to copy their DNA. This process is "replication" in that each strand ofthe original double-stranded DNA molecule serves as template for the reproduction of thecomplementary strand. Hence, following DNA replication, two identical DNA molecules havebeen produced from a single double-stranded DNA molecule. Cellular proofreading and errortoe-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]
• In a cell, DNA replication begins at specific locations in the genome, called "origins".[3]
Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. Inaddition to DNA polymerase, the enzyme that synthesizes the new DNA by addingnucleotides matched to the template strand, a number of other proteins are associated withthe fork and assist in the initiation and continuation of DNA synthesis.
• DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolatedfrom cells, and artificial DNA primers are used to initiate DNA synthesis at knownsequences in a template molecule. The polymerase chain reaction (PCR), a common laboratorytechnique, employs such artificial synthesis in a cyclic manner to amplify a specific targetDNA fragment from a pool of DNA.
Semi-conservative replication, in which the daughter molecules each contain one polynucleotide derived from the original molecule and one newly synthesized strand
Lecture 3: DNA replication
1) Bidirectional replication
2) DNA polymerase
3) How DNA polymerase uses to be bidirectionalreplication
4) DNA topoisomerase
5) DNA polymerase application: PCR andsequencing
Kornberg 의 DNA polymerase
Purified Polymerase
Precipitated insoluble acid product was measured: level of the radioactivity
Substrate must be the tri- and not the diphosphates and only the deoxysugar compounds are active. DNA which must be present may be obtained from animal, plant, bacterial or viral source and the best indications are that all these DNA sample serve equally well in DNA synthesis provided their molecular weight high. The products accumulate until one of the substrates is exhausted and 20 more times greater in amount than the DNA added to the reaction mixture. Inorganic pyrophospate is released in quantities equimolar to the deoxynucleotides converted to DNA
DNA polymerase
• Synthesize new daughter strands of DNA
• An enzyme able to build up a new DNA polynucleotide using anexisting DNA strand as a template called a DNA dependent DNApolymerase
• DNA polymerase synthesize DNA but can also degrade it: Thesequence of the new polynucleotide is dependent on thesequence of the template. DNA polymerization can occur only inthe 5’ 3’ direction
5’-3’ direction of DNA polymerase
• Template-dependent synthesis DNA
• DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide only on to a pre-existing 3'-OH group
• DNA polymerase cannot initiate DNA synthesis unless there is already a short double stranded region to act as a primer
DNA polymerase can degrade polynucleotides as well as synthesize them
• 3’5’ exonuclease,5’3’exonuclease
• Enable a template-dependentDNA polymerase to removenucleotides from the 3’ end ofa strand that it has justsynthesized: Proofreading
Some polymerases are capable of both activities, while others only one or neither of
them • DNA polymerase I: 1957
• DNA polymerase II and III: later found: 1972
• Mutated in DNA pol I still keep replication function II and III found
• DNA polymerase II: mainly proofreading
The replisome assembles at the origin,
Replication fork
Replication origin
Replication Requires a Helicase and a Single-Strand Binding Protein
• Replication requires a helicase to separate the strands of DNA using energy provided by hydrolysis of ATP.
• A single-stranded binding protein is required to maintain the separated strands.
DnaA-ATP binds to short repeated sequences and forms an oligomeric complex that melts DNA.
A hexamer of DnaB forms the replication fork. Helicase and SSB are also required. A short region of A-T-rich DNA is melted.
DnaG is bound to the helicase complex and creates the replication forks. primase – A type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication.
SSBs attach to the unpaired polynucleotides produced by helicase action and prevent the strands from base-pairing with one another or being degraded by nucleases
Initiation: Creating the Replication
Forks at the Origin oriC
• Six DnaC monomersbind each hexamer ofDnaB, and this complexbinds to theorigin.(single strandbinding protein)
Prepriming involves formation of a
complex by sequential association of proteins, which leads to the separation
of DNA strands
DNA Polymerases Are the Enzymes That Make DNA
• DNA is synthesized in both semiconservative replication and DNA
repair reactions.
The DNA polymerase is dependent on the sequence of the template and is determined by complementary base pairing and DNA polymerization can occur only in the 5’ to 3’ direction
DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[5] A DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA.
The primase-helicase complex is used to unwind dsDNA and synthesizes the lagging strand using RNA primers[4] The majority of primers synthesized by primase are two to three nucleotides long.[4]
Once a primer pairs with DNA to be replicated, DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds
DNA polymerases are generally extremely accurate, making less than one error for every 107 nucleotides added. Even so, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a strand in order to correct mismatched bases. If the 5' nucleotide needs to be removed during proofreading, the triphosphate end is lost. Hence, the energy source that usually provides energy to add a new nucleotide is also lost
DNA polymerase I: Repair synthesis replaces a short stretch of one strand of DNA containing a damaged base
DNA is synthesized by adding nucleotides to the 3’–OH end of the growing chain, so that the new chain grows in the 5’ to 3’ direction
Bidirectional Replication?
1967년 Okazaki 가설을 제안
Models for possible structure and reaction in the replicating region of DNA
5’3’ direction od synthesis. No enzymatic mechanism for A If synthesized portion selectively labeled by an extremely short radioactive pulse. IF chromosome replicated discontinuously by one of the mechanisms shown in B, C and D, a larger portion of the radioactive label would be found with unconnected short chain
Because at 20C the generation time(about doubling time of DNA) of E. Coli is about 3 hours and the lysis by T4phage occurs about 140 minutes after infection. Growing cells of E. Coli were exposed to H3-thymidine for various time. DNA was extracted in the denatures state by the NaOH-EDTA treatment and sedimented in alkaline sucrose gradient. (세포의 성장은 KCN 용액을 넣어 세포 증식을 막음, 모든것을 정지 시키고 난후 대장균에서 DNA 추출 후 원심분리) Question ? E. Coli 내에 있는 endonuclease 때문에 초기에 단편 ENA 조작들이 나오는 것이 아닌가?
Endonuclease I-deficient E. Coli.
The initial label of H3-thymidine always appeared in the DNA component with an average sedimentaion
T4 phage-infected E. Coli.
Cells were pulse-labeled after 70 minutes of infection at 20C, when phage DNA is being synthesized actively. After a two-second pulse the radioactive label incorporated was recovered almost exclusively in DNA component with a sedimentaion (top-short fragment), meanwhile after a longer period of labeling, the radioactivity was found in fast sedimental.
A large fraction of the DNA labeled by a short pulse susceptible to degradation by E. Coli exonuclease I
T4 ts A80 and T4 ts B20 30 mutants, which induce temperature-sensitive polynucleotide ligase
Cells were pulse-labeled after 70 minutes of infection at 20C, when phage DNA is being synthesized actively. (첫번째 논문에서 파지가 자라기 시작할때의 시간을 미리 측정 해 놓았음. 20도에서 파지를 대장균에 감염 시킨 후 70분후에 급속하게 파지의 유전자가 증가하게 됨. 아래의 실험은 파지를 높은온도(44도)에서 감염시키고 70분후에 DNA를 추출하기 시작함. 실험에 이동된 파지는 온도를 높이면 Ligase 기능을 잃어버리는 돌연변이 파아지임 : lagging strand 의 Ligase 기능이 없으면 긴 가닥으로 합성되지 않음을 증명함
Okazaki discovered the way in which the lagging strand of DNA is replicated via fragments by conducting an experiment using E. coli. After reacting E. coli with 3[H] Thymidine to synthesize DNA for only ten seconds, he placed the sample in a test tube of alkaline sucrose. The larger, heavier DNA flowed to the bottom of the test tube, while the smaller lighter DNA did not. When samples were taken from the bottom of the test tube, it was found that half were heavy and half were light, proving that half of the DNA was complete and half was in fragments. Then he took a sample of E.coli DNA that had been synthesized for an additional five seconds, and found all the activiy now resulted in the larger molecular weight. Therefore, there were no longer any fragments. This proved that during the five second chase, the RNA primer was removed and the bases were joined together by DNA polymerase I, leaving the new DNA fully mature and repaired.
On the left you see that at very short times of labeling (short pulses) very short pieces of DNA are found (2 sec, 7 sec, 15 sec). However, with longer and longer times, the pieces of DNA get increasing longer (120 sec). He then tried the same experiment with a mutant virus that was defective in a gene called DNA ligase. We will see that this is the enzyme that joins pieces of DNA together into larger structures. In this case (on the right) the labeled pieces of DNA remained short, even after long times of radiolabeling.
Okazaki fragments
RNA primers?
Priming of DNA synthesis
• Replication M13 phage –DNA
• DNA polymerase is involved in M13 phage replication
• However, rifampicin (inhibit RNA polymerase) inhibit replication of M13 phage
• The DNA-degrading enzyme DNase cannot completely destroyed
• Okazaki and his wife to obtained intact primer from in lacked ribonuclease H or nuclease activity of DNA polymerase I. To label only intact primer, they used a capping enzyme that added GMP to the end of RNAs
Since DNA polymerase is incapable of initiating DNA synthesis by itself, it needs a primer to supply a free 3’-end upon which it can build the nascent DNA. Very short pieces of RNA serve this priming function
Prokaryotic DNA polymerases
Pol I: implicated in DNA repair; has 5'->3' (Polymerase) activity and both 3'->5' exonuclease (Proofreading) and 5'->3' exonuclease activity (RNA Primer removal). Pol II: involved in reparation of damaged DNA; has 3'->5' exonuclease activity. Pol III: the main polymerase in bacteria (elongates in DNA replication); has 3'->5' exonuclease proofreading ability.
Arthur Kornberg (March 3, 1918 – October 26, 2007) was an American biochemist who won the Nobel Prize in Physiology or Medicine 1959 for his discovery of "the mechanisms in the biological synthesis of deoxyribonucleic acid (DNA)" together with Dr. Severo Ochoa of New York University
Roger Kornberg was awarded the Nobel Prize in Chemistry in 2006 for his studies of the process by which genetic information from DNA is copied to RNA, "the molecular basis of eukaryotic transcription. RNA polymerase II
Polymerase I (most of replication in E. Coli)
Pol I possesses three enzymatic activities:
A 5' -> 3' (forward) DNA polymerase activity, requiring a 3' primer site and a template strand A 3' -> 5' (reverse) exonuclease activity that mediates proofreading A 5' -> 3' (forward) exonuclease activity mediating nick translation during DNA repair. (fill Okazaki fragment)
DNA Polymerases Have Various Nuclease Activities
• DNA polymerase Ihas a unique 5′–3′exonucleaseactivity that can becombined with DNAsynthesis toperform nicktranslation.
DNA Polymerases Control the Fidelity of Replication
• High-fidelity DNA polymerases involved in replication have a precisely constrained active site that favors binding of Watson–Crick base pairs.
• processivity – The ability of an enzyme to perform multiple catalytic cycles with a single template instead of dissociating after each cycle.
DNA Polymerases Control the Fidelity of Replication
• DNA polymerases oftenhave a 3′–5′ exonucleaseactivity that is used toexcise incorrectly pairedbases.
• The fidelity ofreplication is improved byproofreading
DNA Polymerases Have a Common Structure
• Many DNA polymerases have a large cleft composedof three domains that resemble a hand.
• DNA lies across the “palm” in a groove created by the“fingers” and “thumb.”
Structure from Protein Data Bank 1KFD. L. S. Beese, J. M. Friedman,
and T. A. Steitz, Biochemistry 32 (1993): 14095-14101.
The Two New DNA Strands Have Different Modes of Synthesis
• The DNA polymerase advances continuously when it synthesizes the leading strand (5′–3′), but synthesizes the lagging strand by making short fragments (Okasaki fragments) that are subsequently joined together.
• semidiscontinuous replication – The mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously.
The Two New DNA Strands Have Different Modes of Synthesis
5
3 5
3
Okazaki Fragments Are
Linked by Ligase
• Each Okazaki fragmentstarts with a primerand stops before thenext fragment.
• DNA polymerase Iremoves the primerand replaces it withDNA.
Okazaki Fragments Are Linked by Ligase
• DNA ligase makes the bondthat connects the 3′ end of oneOkazaki fragment to the 5′beginning of the next fragment.
Ligase mutated E.Coli.
DNA polymerase III can synthesize DNA only for a certain distance before it reaches the RNA primer at the 5’ end of the next Okazaki fragment. DNA polymerase III stops and DNA polymerase I come into action, continuing DNA synthesis. DNA ligase synthesize a phosphodiester bond at dissociate position.
Bidirectional Replication
• John Carins experiment in 1960, replication of the E. coli chromosome (두 가닥 을 주형 가닥 으로 사용 used for template)
Tritiated Thymidine
Using autoradiography: incorporating a radioactive label into a substance and then placing the radioactive substance in contact with photographic emulsion so that it can take a picture itself
First generation has one labeled strand (blue). The second round replication, unlabeled parental strand will pick up labeled partner and become singly labeled(A). The labeled parental strand will obtain a labeled partner and become doubly labeled(B). This doubly labeled part should expose the film more and therefore appear darker than rest of the DNA (loop A and C)
http://en.wikipedia.org/wiki/John_Cairns_(biochemist)
Elizabeth Gyurasits and R.B. Wake showed clearly “DNA replication in Bacillus subtilis is
bidirectional.
These investigators’ strategy was to allow B. subtilis cells to grow for a short time in the presence of a weakly radioactive DNA precursor, then for a short time with more strongly radioactive precursor. These short bursts of labeling with a radioactive substance are called pulses of label. (3H-thymidine. Tritium(3H). In the figure the thick labeled showed very strongly at near both forks in bubble. The replication rate is the same in both direction
• At various times between 70 and 120 min, 0.27 ml prewarmed medium containing a higher specific activity of 3H thymidine was added and incubation continued for a further 5, 10 or 20 min (shortly shift from low to hight). Replication was stopped and autordiographs were prepared.
439 types
Type (i)
Type (ii-b)
Type ii -b
Type i-a
Replication 끝나는 시점에 많이 나타남
Linear chromosomal forms
At least two-thirds give a value greater than 0.75, and none is less than 0.5. Considering the degree of uncertainty in fixing the junction between light and heavy regions, there data are consistent with replication processing at approximately the same rate at the two growing points of each loop
Conclusions
• 75% of loops (intact and broken) with two levels of grain density expand bidirectionally
• There is no indication that one of the growing points is slowing down relative to the other as replication proceeds further (복제의 속도는 양방향이 같다)
Replication of the E.coli genome terminates within a defined region
Recognition site called Tus a sequence-specific DNA-binding protein. The six terminator sequences on the E. coli
Topoisomerases release the strain in replicating circular DNAs
Cairns recognized a “swivel” in the DNA duplex that would allow the DNA strands on either side to rotate to relieve the strain
Topoisomerase I and II single strand break and both strand break and reseal.
• http://www.youtube.com/watch?v=EYGrElVyHnU
• http://www.youtube.com/watch?v=Jw2rltYV1yo&NR=1&feature=endscreen
