Functions of DNA

1. Deepa John Harini Chandra Affiliations Functions of DNA DNA, which carries the blueprint of life, is the key performer of the central dogma. In addition to transmitting hereditary information from one generation to the next by means of replication, the genes of DNA code for protein sequences in all organisms.

2. Master Layout (Part 1) 1 This animation consists of 3 parts: Part 1 – Proposed models for DNA replication Part 2 – Replication of DNA Part 3 – Transcription of DNA Parent DNA molecule 2 Replication 3 Semi-conservative model Conservative model Dispersive model 4 5 Daughter DNA molecules

3. Definitions of the components:Part 1 – Proposed models for DNA replication 1 1. Parent DNA molecule: The DNA molecule that is being replicated to produce new daughter DNA molecules. 2. Replication: It is a fundamental process that occurs in all living organisms to transmit their genetic material from one generation to the next. Two copies of nucleic acid are synthesized from one parent molecule during the process of cell division such that each daughter cell obtains one copy of the genetic material. The process can be inhibited by novobiocin, nalidixic acid and coumeomycin in prokaryotes and by aphidicolin, N-ethylmaliamide and camptothucin in eukaryotes. 3. Daughter DNA molecules: The new DNA molecules that are synthesized from the parental DNA molecules and are complementary to parental DNA molecules. 4. Conservative model: One of the three proposed models for DNA replication. According to the conservative model, the two parental strands of DNA as a whole serve as a template for the synthesis of progeny DNA molecules. Thus, one of the daughter DNA molecules is actually the parental DNA while the other daughter DNA consists of two newly synthesized strands from fresh nucleotides. 2 3 4 5

4. Definitions of the components:Part 1 – Proposed models for DNA replication 1 5. Dispersive model: One of the three proposed models for DNA replication. The dispersive model of DNA replication hypothesizes that the parental DNA molecule is cleaved into smaller double stranded DNA segments which serve as the template for synthesis of new DNA strands. The segments then reassemble into complete DNA double helices, with parental and daughter DNA segments interspersed. 6. Semi-conservative model: One of the three proposed models for DNA replication. According to the semi-conservative model of replication, each parental strand acts as a template for the synthesis of a new strand of DNA which is complementary to the parental strand. Each daughter DNA molecule always has one parental DNA strand and one newly synthesized daughter strand. This model was ultimately proved by an experiment conducted by Meselson & Stahl. 2 3 4 5

5. Part 1, Step 1: 1 Conservative model: Original parent DNA molecule Round 1 replication 2 First generation daughter molecules New nucleotides 3 Round 2 replication Second generation daughter molecules 4 Audio Narration Action Description of the action Several models have been postulated to explain the process of DNA replication. According to the conservative model, the two parental strands of DNA as a whole serve as a template for the synthesis of progeny DNA molecules. Thus, one of the daughter DNA molecules is actually the parental DNA while the other daughter DNA consists of two newly synthesized strands from fresh nucleotides. (Please use black background) First show the green strands on top with its label. Next show the two arrows below it and the red & green figures below. Next show the down arrows and the figures below this with text as depicted in the animation. As shown in animation 5

6. Part 1, Step 2: 1 Dispersive model: Original parent DNA molecule Round 1 replication 2 First generation daughter molecules 3 Round 2 replication Second generation daughter molecules 4 Audio Narration Action Description of the action The dispersive model of DNA replication hypothesizes that the parental DNA molecule is cleaved into smaller double stranded DNA segments which serve as the template for synthesis of new DNA strands. The segments then reassemble into complete DNA double helices, with parental and daughter DNA segments interspersed. The content of parental DNA in the double helix goes on decreasing with each generation. (Please use black background) First show the green strands on top with its label. Next show the two arrows below it and the red & green figures below where the green and red strands must be interspersed. Next show the down arrows and the figures below this with text as depicted in the animation. As shown in animation 5

7. Part 1, Step 3: 1 Semi-conservative model: Original parent DNA molecule Round 1 replication 2 First generation daughter molecules Newly synthesized DNA strand Parent DNA strand 3 Round 2 replication Second generation daughter molecules 4 Audio Narration Action Description of the action According to the semi-conservative model of replication, each parental strand acts as a template for the synthesis of a new strand of DNA which is complementary to the parental strand. Each daughter DNA molecule always has one parental DNA strand and one newly synthesized daughter strand. (Please use black background) First show the green strands on top with its label. Next show the two arrows below followed by the figures below with the green and red strands intertwined. Next, the arrows below must be shown followed by figures below. As shown in animation 5

8. Part 1, Step 4: 1 Meselson & Stahl Experiment - proof for the semi-conservative model of repllication Cells transferred and grown in normal 14N medium E.Coli grown in 15N containing medium 2 Round 1 replication Round 2 replication First generation daughter molecules Second generation daughter molecules 3 Heavy 15N labeled parent DNA 15N-14N labeled hybrid DNA 15N-14N hybrid DNA + 14N light DNA 4 Centrifugation pattern in CsCl density gradient Audio Narration Action Description of the action Of the three replication models suggested, Meselson and Stahl proved that the semiconservative model was correct. For this they grew E.coli cultures for several generations in 15N-containing medium so that the bases in DNA contained 15N instead of 14N. Next they transferred & grew the cultures for several generation in an 14N-containing medium. Throughout the period of growth, samples were taken, cells lysed and the DNA analyzed by centrifugation in CsCl gradient. The parent DNA showed 1 band in CsCl gradient corresponding to 15N DNA, the 1st generation daughter molecules also showed 1 band which was not at the same position as parent DNA. This corresponded to 14N-15N DNA while the 2nd generation showed 2 bands, one of 14N-15N and the other of 14N light DNA. These results exactly matched the semiconservative replication model . First show the figure on top left with the light pink cylindrical shapes. This must be zoomed into and the green strands below must be shown. After zooming out, the figure below that must be shown. Next, the pink cylindrical shapes must be transferred into the light blue solution in the flask. The number of pink shapes must increase by a few numbers after which one of the shapes must be zoomed into and the red & green strands shown followed by the figure below that. Next, the number of pink cylindrical shapes must again increase by 4-5 and then one must be zoomed into and the last figure strands must be shown followed by the figure on the bottom. As shown in animation 5

9. Master Layout (Part 2) 1 This animation consists of 3 parts: Part 1 – Proposed models for DNA replication Part 2 – Replication of DNA Part 3 – Transcription of DNA 2 Replication fork Single strand binding protein Helicase 3 RNA primer Primase Template DNA DNA pol III 4 DNA pol I DNA ligase Lagging strand 5

10. Definitions of the components:Part 2 – Replication of DNA 1 1.Template: A polynucleotide DNA strand that serves as the guide for making a complementary polynucleotide. 2.Origin of replication: Unique sequences in the genome where replication is initiated. 3.Replication fork: The point where the two parental DNA strands separate to allow replication. 4. Helicase: An enzyme that unwinds a polynucleotide double helix using energy derived form ATP hydrolysis. 5.Single strand binding protein (SSB): Binds to single stranded DNA during replication and keeps it from base pairing with a complementary strand. 6. Primer: A small RNA fragment that provides the free 3’ OH end needed for DNA replication to begin. 7. Primase: The enzyme that catalyzes the synthesis of a small piece of RNA complementary to the single stranded DNA that provides the free 3’ OH end needed for DNA replication to begin. It is a key component because DNA polymerases cannot initiate the synthesis of DNA without an RNA primer. 2 3 4 5

11. Definitions of the components:Part 2 – Replication of DNA 1 8. Supercoil: A form of circular double stranded DNA in which the double helix coils around itself like twisted rubber band, leading to a lot of torsional strain. 9. DNA polymerase: An enzyme that synthesizes DNA by linking together deoxyribonucleoside monophosphates in the order directed by the complementary sequences of nucleotides in a template strand. DNA polymerases can synthesize DNA only in 5’ to 3’ direction and can add nucleotides only on to a pre-existing 3'-OH group. Prokaryotes have three types of DNA Polymerase while eukaryotes have five. 10. Leading and lagging strands:DNA polymerase can synthesize DNA only in 5’to 3’ direction. Therefore, it synthesizes one strand (leading strand) continuously and the other strand(lagging strand) discontinuously. Each new piece synthesized on the lagging strand template is called Okazaki fragment. 11. DNA ligase: This enzyme catalyze the formation of a phosphodiester bond between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the another thereby covalently linking DNA fragments together during DNA replication and repair. 2 3 4 5

12. Part 2, Step 1: 1 Unwinding the DNA double helix 3’ 3’ Single Stranded Binding (SSB) proteins 5’ 5’ Direction of fork movement 2 DNA helicase DNA gyrase 5’ SSBs stabilize the ssDNA 3 3’ 5’ 3’ DNA gyrase 4 Audio Narration Action Description of the action The transparent blue oval must move across the intertwined strands & separate them (Please use black background) First show the green & red strands on top. Next show the blue transparent oval and the yellow circles. The blue oval must then move in the direction indicated. As it moves the two strands must be separated as shown below and the yellow circles must coat the red strand on either side. Simultaneously, the purple ‘pie shape’ must move in the same direction along the two strands. DNA undergoes semi-conservative, bi-directional replication which begins with the unwinding of the DNA double helix. This is done by the enzyme DNA helicase which binds to the replication fork and unwinds the DNA using the energy of ATP hydrolysis. As this occurs, the enzyme DNA gyrase relieves the trosional strain that builds up during the process in the unwound part of the double helix. The single-stranded binding proteins bind to and stabilize the unwound single stranded regions of the DNA helix to allow replication to occur. 5

13. Part 2, Step 2: 1 Initiation 5’ 2 Lagging strand 5’ RNA primers 5’ 3 3’ Leading strand 5’ 3’ Primase 4 Audio Narration Action Description of the action As shown in animation. Initiation of DNA replication is carried out by a primase enzyme which synthesizes short RNA primer fragments since DNA Polymerase is not capable of carrying out this process. The SSBs are displaced as the short fragments get synthesized. Synthesis takes place in the 5’ to 3’ direction such that nucleotides can be added to the free 3’ OH group with concomitant cleavage of the high energy phosphate bond of the incoming nucleotide. . (Please use black background) First show the figure from the previous step as such followed by the pink circles. These must move in the directions shown in the animation and as they move on the top strand, the yellow circles must be displaced. Once they move a small distance, the small blue fragment must appear. 5

14. Part 2, Step 3: 1 Elongation Lagging strand Okazaki fragment 5’ 5’ 5’ 2 5’ 5’ Newly synthesized complementary DNA Leading strand DNA Polymerase III 5’ 5’ 3 Next Okazaki fragment 3’ 5’ 3’ 3’ 5’ 4 3’ Audio Narration Action Description of the action As shown in animation. Elongation takes place continuously in the 5’-3’ direction on one strand, known as the leading strand. On the other strand, replication is discontinuous with short primers being added as the helicase unwinds the double helix. Elongation is carried out by DNA Pol III, a highly processive enzyme. The short fragments synthesized on the lagging strand are known as Okazaki fragments. (Please use black background) First show the figure on top upto the stage depicted in previous slide. Next show the green ovals which move in the direction indicated with the yellow circles on top getting displaced as the oval on top moves towards it. Once it moves the distance indicated, the red and green fragments must appear. Next the blue oval and violet pie shaped object must move as in step 1 with unwinding of the green & red strands as shown below. Again yellow circles must coat the red strand. Then the pink circle must appear and move as shown with appearance of the small blue fragment. This is followed by the green ovals moving with appearance of the green & red fragments as shown in animation. 5

15. Part 2, Step 4: 1 Removal of primers & sealing gaps Lagging strand DNA Polymerase I Okazaki fragments 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 2 3’ Leading strand 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3 DNA ligase Fully replicated DNA strands 4 Audio Narration Action Description of the action As shown in animation. (Please use black background) First show the red and green strands with their small blue fragments. Next show the grey pie-shaped objects moving across these blue fragments as shown in animation, after which they must be removed and filled with green and red fragments of the same size. The gaps remaining in the top green strand must then be filled in by the violet oval shaped objects which must be move across the gaps after which they must be filled. The entire DNA is unwound in this manner by DNA helicase with DNA Pol III synthesizing the new complementary strands. The RNA primers are then removed and the gaps filled by the enzyme DNA Pol I. The Okazaki fragments on the lagging strand, which still have a nick between two consecutive fragments, are then joined together by means of the enzyme DNA ligase. Sealing of the nicks completes the process of replication after which all the machinery dissociates from the DNA strands. 5

16. Master Layout (Part 3) 1 This animation consists of 3 parts: Part 1 – Proposed models for DNA replication Part 2 – Replication of DNA Part 3 – Transcription of DNA RNA polymerase 2 Rewinding Unwinding 3 Movement of polymerase 5' 4 5 Source : Biochemistry by Stryer, 6th edition (ebook):

17. Definitions of the components:Part 3 – Transcription of DNA 1 1.Transcription:Transcription is a process by which information from a double stranded DNA molecule is converted to a single stranded RNA molecule by making use of one strand as the template. The process differs slightly between prokaryotes and eukaryotes and is inhibited by rifampicin in prokaryotes, by a-aminitin in eukaryotes and by acridine and actinomycin D in both. 2. RNA polymerase: An enzyme that catalyzes the synthesis of RNA molecules from a DNA template. In prokaryotes, the RNA polymerase holoenzyme consists of the core enzyme form which has four polypeptides(two α,β,β‘) bound to another factor called sigma factor. In eukaryotes, three RNA polymerases exist called RNA pol I, RNA pol II and RNA pol III. RNA pol II is involved in the transcription process. RNA Pol IV, found in plants, is involved in the synthesis of small interfering RNA (siRNA) which is responsible for degradation of homologous mRNA for gene silencing. 3. Nascent RNA - The RNA molecule that is being synthesized from DNA and in the process of development in order to produce the final mature RNA. 4. Coding strand – The DNA strand that has same base sequence as that of the RNA transcript being produced. In other words, it is the complementary sequence of the template strand. 5. Template strand – The DNA strand of a gene that serves as a template for the synthesis of RNA strand during transcription. It is complementary to that of the RNA transcript that is produced. 6. Promoter: A specific recognition nucleotide sequence in DNA to which the RNA polymerase binds for initiation of transcription. 2 3 4 5

18. Definitions of the components:Part 3 – Transcription of DNA 1 7. Closed promoter complex: The complex formed by relatively loose binding between RNA polymerase and the promoter region. It is said to be closed because the DNA duplex remains intact and there is no melting of DNA base pairs. 8. Open promoter complex: The complex formed by tight binding of RNA polymerase with the promoter element. It is said to be open because approximately 17 base pairs of DNA open up. 9. Sigma factor: A prokaryotic transcription initiation factor which is essential for promoter recognition. If the sigma factor is not present, the core enzyme binds to DNA in various places but does not initiate transcription efficiently from any one of them. 10. Transcription factors: Specific proteins that are required for the initiation of transcription by RNA polymerases. In prokaryotes it is the sigma factor. In eukaryotes they are TFIID, TFIIB, TFIIH, TFIIE and TFIIF. 11. Terminator sequence: A transcription regulatory sequence located at the distal end of a gene that signals the termination of transcription. 12. Rho factor: Rho factor is a protein essential for prokaryotic transcription termination. It has two domains, one that binds to ATP and the other that binds to the newly synthesized RNA transcript. 13. rut elements: Rho utilization elements. These are the sequences to which rho protein binds and are essential for its terminator function. 2 3 4 5

19. Part 3,Step 1: 1 Prokaryotic transcription initiation s factor Closed promoter complex 3' 5' 5' 3' 2 Promoter RNA polymerase Open promoter complex 3 5' 3' 5' 3' 4 Action Description of the action Audio Narration Transcription is the process by which information from a double stranded DNA molecule is converted to a single stranded RNA molecule. For prokaryotic transcription to begin, the RNA polymerase holoenzyme consisting of the core enzyme bound to the σ factor must bind to the promoter region. The s factor is responsible for recognition of the promoter sequence. Binding results in a local unwinding of around 17 base pairs centered around the promoter. At this point RNA polymerase is correctly oriented to begin transcription from the +1 nucleotide. (Please use black background & redraw all figures) First show the text box with prokaryotic transcription initiation. Then show the two strands on top. Next show the blue shape with brown C shape inside it coming in from left and occupying the red area. Then show the blue & brown shapes moving across the red region. When this happens, the two strands must separate in the red region to give rise to the figure below. As shown in the animation 5 Source:  Molecular Biology of the Cell 5/e Garland Science, 2008

20. Part 3,Step 2: 1 Eukaryotic transcription initiation Promoter Closed promoter complex 3' 5' 5' 3' 2 TFIIB TFIIB TFIIF TFIIF TFIID TFIID RNA polymerase II Transcription factors Open promoter complex TFIIE TFIIE 3 5' 3' TFIIH TFIIH 3' 5' 4 Action Description of the action Audio Narration As shown in the animation (Please use black background & redraw all figures) First show the text box with eukaryotic transcription initiation. Then show the two strands on top. Next show the yellow oval with orange circles inside it coming in from left and occupying the red area. Then show the circle moving across the red region. As it moves, the two strands must separate out and give rise to the figure shown below. In case of eukaryotic transcription initiation, RNA polymerase binds to the promoter region along with several transcription factors (TF), which recognize the promoter site. The first step is the binding of TFIID. This complex acts as a binding site for TFIIB which then recruits RNA polymerase II and TFIIF. Finally TFIIE and TFIIH also bind to produce the complete transcription initiation complex. 5 Source:  Molecular Biology of the Cell 5/e Garland Science, 2008

21. Part 3,Step 3: 1 Elongation Direction of movement 2 Template DNA strand Newly synthesized RNA Promoter clearance - s factor dissociates Helix unwinding 3 Helix re-winding 5' 5' 3' 3' 5' 5' 3' 3' Terminator sequence 4 RNA transcript Audio Narration Action Description of the action In prokaryotes, the sigma factor dissociates from the core enzyme, a process known as promoter clearance, once it has synthesized around 9-10 nucleotides. RNA polymerase continues elongation of new RNA chain in the 5’-3’ direction by unwinding the DNA ahead of it as it moves and re-winding the DNA helix that has already been transcribed. Eukaryotic elongation is similar except that the polymerase involved is RNA polymerase II. (Please use black background & redraw all figures) First show the strands on top with the red region separated and the yellow oval & violet triangle bound to it. This complex must then move across the red region in the direction shown. As it moves, the blue strand above it must appear. Once it crosses the red region, the violet triangle must dissociate. The yellow oval must continue to move in the same direction. As it moves, the black strands in front must open up in a manner similar to the red & the strands that the yellow oval crosses must rejoin again. As it moves, more blue strand must be formed and must come out in a manner shown in the figure below. This continues until it reaches the green region of the strands. As shown in the animation 5 Source:  Molecular Biology of the Cell 5/e Garland Science, 2008

22. Part 3, Step 4: 1 Termination: Rho dependent 5' 3' 2 5' 3' Stalled transcription complex Release of RNA transcript ATP+ H2O ADP + Pi AC rich rut element 3 RNA transcript Rho protein RNA polymerase dissociates 4 Action Description of the action Audio Narration Termination of transcription is signaled by controlling elements called terminators that have specific distinguishing features. Prokaryotic termination can be rho-dependent or rho-independent. In rho-dependent termination, one subunit of the rho protein gets activated by binding to ATP after which the other subunit binds to the RNA transcript and moves to the stalled transcription complex. Hydrolysis of ATP leads to release of the RNA transcript as well as RNA Polymerase, thereby terminating the transcription process. (Please use black background & redraw all figures) First show the strands which are unwound at the green marked region with the yellow oval and blue strand bound to it but not moving. Then show the violet ovals binding to the blue strand as shown and moving to the yellow oval. Next show the equation given, following which the yellow circle, blue strand as well as the purple ovals must all dissociate from the black strands. The purple ovals must be last to dissociate. As shown in the animation 5 Source:  Molecular Biology of the Cell 5/e Garland Science, 2008

23. Part 3, Step 5: 1 Termination: Rho independent 5' 3' 2 5' 3' Stalled transcription complex Self complementary region forming hair pin loop Conserved AAA residues in template 3 Release of RNA transcript RNA polymerase dissociates 4 Action Description of the action Audio Narration (Please use black background & redraw all figures) The blue strand which is comes out as the yellow oval moves towards the right, must be made to assume this shape once the yellow oval reaches the green region of the figure. Once the blue strand assumes this shape, both the yellow oval and blue strand must dissociate from the black strands above. Rho-independent termination takes place due to the formation of a hairpin loop structure by the newly synthesized RNA transcript. The terminators for this mechanism have two specific features – the first is a region on the template that will produce a self-complementary sequence on the RNA transcript located around 15-20 nucleotides before the expected end of the RNA. The next feature is a conserved sequence of 3 adenine residues on the template near the 3’ end of the hairpin. Formation of the hairpin disrupts the weak AU interactions, thereby allowing dissociation of the newly synthesized RNA transcript and the RNA polymerase. As shown in the animation 5 Source:  Molecular Biology of the Cell 5/e Garland Science, 2008

24. Interactivity option 1:Step No:1 1 Nitrous acid deaminates cytosine to produce uracil, a base that pairs with adenine. After this conversion, which base pair occupies this position in each of the daughter strands resulting from one round of replication and two rounds of replication? 2 A) After one round of replication, two GC and after two rounds, two GC and two AT B) After one round of replication, one GC, one AT and after two rounds, two AT C)After one round of replication, one GC, one AU and after two rounds, two GC and one AU, one AT. 3 D) After one round of replication, two GC and after two rounds, two GC and one AU, one AT. 4 Results Interacativity Type Options Boundary/limits User has to choose one of the four options. If A or B or D chosen, then they must turn red. User can however continue till he gets the right answer(C) which must turn green. User is then directed to step 2. User has to choose one of the four options. If A or B or D chosen, then they must turn red. User can however continue till he gets the right answer(C) which must turn green. User is then directed to step 2. Choose the correct option 5

25. Interactivity option 1:Step No:2(a) Nitrous acid C U G Original Base pair Modified Base pair Replication round 1 U C A G Replication round 2 C C T U G G A A

26. Interactivity option 2:Step No:1 1 FRET (Fluorescence Resonance Energy Transfer) is a technique for measuring interactions between two proteins in vivo. In this technique, two different fluorescent molecules (fluorophores) are genetically fused to the two proteins of interest.In FRET, light energy is added at the excitation frequency for the donor fluorophore, which transfers some of this energy to the acceptor, which then re-emits the light at its own emission wavelength.The efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation. Using this technique it is possible to show that sigma factor dissociates from RNA polymerase during elongation of prokaryotic transcription process. Click on the button below to view the process. 2 3 4 Results Interacativity Type Options Boundary/limits User has to click on the action button below and the user is then directed to step 2. User has to click on the action button below and the user is then directed to step 2. Choose the action button 5

27. Interactivity option 2:Step No:2 Open promoter complex – ready for elongation s s Fluorescent acceptor Bright fluorescence due to proximity of donor & acceptor fluorophores Fluorescent donor s factor RNA polymerase 5' 5' 3' 3' 5' 5' 3' 3' Direction of movement Template DNA strand Newly synthesized RNA Decrease in fluorescence intensity as donor moves away from acceptor Promoter clearance - s factor dissociates

28. Questionnaire 1 1. The unwinding of DNA is done by _____. Answers: a) DNA polymerase b) DNA repair enzymes c) DNA helicase d) ‏DNA ligase 2. Replication forks are characteristic of the DNA replication of _____. Answers: a) Viruses b) Prokaryotes c) Eukaryotes d)‏ Both viruses and eukaryotes 3. Rifampicin is an inhibitor of transcription of bacteria. It binds to the following subunit of RNA polymerase. Answers: a) βb) β' c) α d)‏σ 4. Okazaki fragments are characteristic of the DNA replication of the _____ strand. Answers: a) Leading strand b) Lagging strand c) Both a and b d) Non of the above‏ 5. α- amanitin at very low concentrations inhibit Answers: a) RNA pol II only b) RNA pol I only c) RNA pol III only d)‏ Both RNA pol I and RNA pol II 2 3 4 5

29. Links for further reading Books: Molecular biology by Robert F. Weaver, 4th edition Genetics by Peter J. Russel, 5th edition Biochemistry by Stryer, 6th edition