6 MPBELL BIOLOY ENH EDIION Reece Urry ain Wasserman Minorsky Jackson DN: he Molecular Basis of Inheritance DN Deoxyribonucleic acid DN he blueprint to making proteins!!! hromosomes located inside the nucleus contains long coiled s of DN Lecture Presentation by Dr Burns NV Biol 20 opyright 204 Pearson 2009 Education, Pearson Education, Inc. Inc. DN s Discovery Watson and rick Rosalind Franklin he Players rick: Ph.D. student at ambridge in England working on X-ray rystallography of the protein hemoglobin Watson: Young merican scientist visiting the lab to do some work on a protein Both were interested in unraveling the secret of DN s structure it was not what they were supposed to be working on Wilkins: Working on DN structure, crystallized DN fibers Franklin: Working at the same university as Wilkins, just down the hall. Did the X-ray rystallography on Wilkins DN fibers Linus Pauling: discovered the three dimensional structure of proteins know as alpha helixes hargaff: Discovered that = and = denine levels always equal thymine levels, uanine levels always equal cytosine
Franklin gave a talk describing her work with the X-Ray rystallography, Watson attended but he was not the crystallographer and did not see the implications of her work Watson and rick met with Wilkins and he shared Franklin s work with both of them (without her permission or knowledge) Watson and rick put all the pieces of information together. hey built models to help them come up with the structure. hey knew it was a race so they published a one page article in Nature with their ideas they performed no experiments but were able to see the big picture rick, Watson and Wilkins received the Nobel Prize for their work. Rosalind received no credit until much later. She died before the Nobel Prize, the prize is not awarded after a person has died Figure 6.7 5 end Hydrogen bond 3 end 3.4 nm nm (a) Key features of DN structure 0.34 nm 3 end (b) Partial chemical structure 5 end (c) Space-filling model nimation: Hershey-hase Experiment Right-click slide / select Play 204 Pearson Education, Inc. Fig. 4.4 Nucleotide Structure Figure 6.5 Sugar phosphate backbone end Nitrogenous bases hymine () denine () ytosine () Phosphate uanine () Sugar (deoxyribose) DN nucleotide end Nitrogenous base 2
DN Structure Fig. 4.3- Nucleotides that build DN have: One phosphate (P has three) One sugar = deoxyribose One base. he nucleotides vary in the type of base there are four different bases in DN: denine (), hymine (), uanine (), ytosine () here is a 5 end and a 3 end 3
nimation: DN and RN Structure Rightclick slide / select Play Bonds he sugars and phosphates link together by covalent bonds to form the rail on the outside = phosphodiester linkage. he sugars are covalently bound to a base he complementary bases are attracted to each other by hydrogen bonds Double Helix wo s bonded together by hydrogen bonds between the bases = weak bonds Each has nucleotides bonded together covalently by the phosphate and the sugar Base pairs are two nucleotides, one on each complementary of a DN molecule Base Pairs he bases pair up in a specific manner: denine () pairs with hymine () uanine () pairs with ytosine () Purines: denine and uanine Pyrimidines: hymine and ytosine 4
Figure 6.8 Sugar Sugar denine () Sugar hymine () Sugar Remember that on one : he base is covalently bonded to the sugar, which is covalently bonded to the phosphate Between the two s the bases are bonded together by hydrogen bond uanine () ytosine () he bonds between the sugars and phosphates are he bonds between the bases are. Peptide 2. Phosphodiester 3. Hydrogen 4. Ionic 25% 25% 25% 25%. Peptide 2. Phosphodiester 3. Hydrogen 4. Ionic 25% 25% 25% 25% Peptide Phosphodiester Hydrogen Ionic Peptide Phosphodiester Hydrogen Ionic denine pairs with uanine pairs with. hymine 2. uanine 3. ytosine 33% 33% 33%. hymine 2. denine 3. ytosine 33% 33% 33% hymine uanine ytosine hymine denine ytosine 5
he bases are bound to he bases are bound to the sugar by this kind of bond. Sugars 2. Phosphates 50% 50%. ovalent 2. Phosphodiester 3. Hydrogen 4. Ionic 25% 25% 25% 25% Sugars Phosphates ovalent Phosphodiester Hydrogen Ionic he sugar in DN is DN replication. Ribose 2. Deoxyribose 3. lucose 4. ellulose 25% 25% 25% 25% he relationship between structure and function is manifest in the double helix Watson and rick noted that the specific base pairing suggested a possible copying mechanism for genetic material Ribose Deoxyribose lucose ellulose DN Replication When the structure of DN was worked out it became apparent how it happens It is semiconservative replication Each of DN is the template for building new complementary s he Basic Principle: Base Pairing to a emplate Strand Since the two s of DN are complementary, each acts as a template for building a new in replication In DN replication, the parent molecule unwinds, and two new daughter s are built based on base-pairing rules 6
nimation: DN Replication Overview Right-click slide / select Play Figure 6.9- Figure 6.9-2 (a) Parent molecule (a) Parent molecule (b) Separation of s Figure 6.9-3 Semiconservative model Watson and rick s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old (derived or conserved from the parent molecule) and one newly made (a) Parent molecule (b) Separation of s (c) Daughter DN molecules, each consisting of one parental and one new ompeting models were the conservative model (the two parent s rejoin) and the dispersive model (each is a mix of old and new) 7
Figure 6.0 (a) onservative model Parent cell First replication Second replication Fig. 4.3 (b) Semiconservative model (c) Dispersive model DN Replication: loser Look BioFlix: DN Replication he copying of DN is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DN replication etting Started Replication begins at particular sites called origins of replication, where the two DN s are separated, opening up a replication bubble eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied t the end of each replication bubble is a replication fork, a Y-shaped region where new DN s are elongating nimation: Origins of Replication Right-click slide / select Play 8
0.5 m 0.25 m Figure 6.2 (a) Origin of replication in an E. coli cell Origin of replication Doubleed DN molecule wo daughter DN molecules Parental (template) Replication bubble Daughter (new) Replication fork (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) Bubble Double-ed DN molecule Daughter (new) Replication fork wo daughter DN molecules DN DN Replication. n enzyme, helicase, unwinds the DN molecule and breaks the hydrogen bonds between the base pairs 2. Single- binding proteins bind to each and keep them from reforming the double helix 3. opoisomerases produce breaks in the DN molecule to relieve the stress of unwinding, then they also repair these breaks. Figure 6.3 DN Replication opoisomerase Helicase Primase Single- binding proteins RN primer Now the complementary needs to be built: 4. Enzymes called DN polymerases build the new complementary by adding new nucleotides to the 3 end which pair with the old DN. 5. But DN polymerase can not start the process. primer of RN bases is first built for the complementary. 6. n enzyme called primase adds the RN bases, then DN polymerase can take over and keep building the complementary. 7. he primer is replaced by DN bases DN DN Replication DN DN Replication 8. DN polymerase builds the new complementary from the 5 end to the 3, by adding the nucleotides to the 3 end = leading But the other also needs to be replicated but it can only build new s by adding to the 3 end 9. he other = lagging, is build in short stretches going from 5 to 3 0. he short s being built are called Okazaki fragments. DN ligase join the Okazaki fragments 9
Synthesizing a New DN Strand Enzymes called DN polymerases catalyze the elongation of new DN at a replication fork Most DN polymerases require a primer and a DN template he rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Energy to power building complementary he incoming nucleotides have three phosphates, only one is used to bond to the sugar molecule he energy needed to build the new DN comes from taking the other two phosphates off. Figure 6.4 New emplate ntiparallel Elongation Sugar Phosphate Base he antiparallel structure of the double helix affects replication OH DN polymerase DN polymerases add nucleotides only to the free end of a growing ; therefore, a new DN can elongate only in the to direction P P i Pyrophosphate OH Nucleoside triphosphate 2 P i ntiparallel Elongation long one template of DN, the DN polymerase synthesizes a leading continuously, moving toward the replication fork nimation: Leading Strand Rightclick slide / select Play 0
ntiparallel Elongation o elongate the other new, called the lagging, DN polymerase must work in the direction away from the replication fork he lagging is synthesized as a series of segments called Okazaki fragments, which are joined together by DN ligase nimation: Lagging Strand Rightclick slide / select Play Figure 6.6a Figure 6.6b- emplate Leading Overview Origin of replication Lagging Lagging 2 Overall directions of replication Leading Figure 6.6b-2 Figure 6.6b-3 emplate RN primer for fragment emplate RN primer for fragment Okazaki fragment
Figure 6.6b-4 Figure 6.6b-5 emplate RN primer for fragment emplate RN primer for fragment RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment 2 RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment 2 2 Figure 6.6b-6 Figure 6.7 emplate RN primer for fragment Leading Overview Origin of replication Lagging RN primer for fragment 2 Okazaki fragment 2 Okazaki fragment 2 2 Parental DN DN pol III Primer Primase Leading DN pol III 4 Leading Lagging Overall directions of replication Lagging DN pol I DN ligase 3 2 2 Overall direction of replication Figure 6.7a Leading Overview Origin of replication Lagging Figure 6.7b Leading Overview Origin of replication Lagging Lagging Overall directions of replication Leading Leading Lagging Overall directions of replication Leading Parental DN Leading DN pol III Primer Primase Primer DN pol III Lagging 4 DN pol I DN ligase 3 2 2
he DN Replication omplex he proteins that participate in DN replication form a large complex, a DN replication machine he DN replication machine may be stationary during the replication process Recent studies support a model in which DN polymerase molecules reel in parental DN and extrude newly made daughter DN molecules nimation: DN Replication Review Right-click slide / select Play Figure 6.8 Parental DN DN pol III Leading Youube - DN Replication Process Youube - DN Replication (Very realistic 3D animation) onnecting protein Helicase DN pol III Lagging Lagging template Replicating the Ends of DN Molecules Limitations of DN polymerase create problems for the linear DN of eukaryotic chromosomes Replication at end of DN t the end of the DN a small portion of the is not replicated he usual replication machinery provides no way to complete the ends, so repeated rounds of replication produce shorter DN molecules with uneven ends his is not a problem for prokaryotes, most of which have circular chromosomes So we don t lose important genetic information, DN s have non-coding end caps hese end caps are called telomeres 3
Figure 6.20 Ends of parental DN s Lagging Last fragment RN primer Leading Lagging Next-to-last fragment Figure 6.20a Ends of parental DN s Leading Lagging Parental Removal of primers and replacement with DN where a end is available Lagging Last fragment RN primer Next-to-last fragment New leading New lagging Second round of replication Parental Removal of primers and replacement with DN where a end is available Further rounds of replication Shorter and shorter daughter molecules Figure 6.20b elomeres Second round of replication Eukaryotic chromosomal DN molecules have special nucleotide sequences at their ends called telomeres New leading New lagging hey postpone the erosion of genes near the ends of DN molecules Further rounds of replication It has been proposed that the shortening of telomeres is connected to aging Shorter and shorter daughter molecules Figure 6.2 Replication at end of DN elomerase build the telomeres. Embryos have high telomerase activity, as you age you lose this activity. ancer cells have telomerase activity m 4
Fig. 4.24- Fig. 4.24-2 Mistakes repair mechanisms Before a cell can divide, it must make a complete copy of itself here are millions of bases that need to be added to the DN s many chances for something to go wrong Enzymes will take out the wrong nucleotide and replace it with the correct one auses of Mutations Random error sometimes things just go wrong. Mutagens chemicals that damage the DN and cause mutations in replication igarette smoke Sunlight Many chemicals (benzene) Results of Mutations few things can happen if DN mutates before the cell replicates: Enzymes can repair the damage Or he cell may commit suicide (apoptosis) Or he cell may replicate and the mutation becomes permanent Proofreading and Repairing DN In nucleotide excision repair, an endonuclease cuts out and DN polymerase replaces damaged stretches of DN 5
Repair Mechanisms Fig. 4.25- Photorepair UV light can cause pyrimidines dimers to occur. Photolyase uses visible light to break dimer Nucleotide excision repair mismatched pairs are recognized and removed an endonuclease cuts out and DN polymerase replaces damaged stretches of DN then DN ligase joins the segements Fig. 4.25-2 Figure 6.9 Nuclease DN polymerase DN ligase Evolutionary Significance of ltered DN Nucleotides Which enzyme unwinds the DN molecule and breaks the hydrogen bonds between the base pairs? Error rate after proofreading repair is low but not zero Sequence changes may become permanent and can be passed on to the next generation hese changes (mutations) are the source of the genetic variation upon which natural selection operates. helicase 2. opoisomerases 3. DN polymerases 4. Primase 5. DN ligase 20% 20% 20% 20% 20% 2 3 4 5 opyright 2009 Pearson Education, Inc. 6
Which enzyme produces breaks in the DN molecule to relieve the stress of unwinding, then they also repair these breaks? Which enzyme builds the new complementary by adding new nucleotides to the 3 end which pair with the old DN?. helicase 2. opoisomerases 3. DN polymerases 4. Primase 5. DN ligase 20% 20% 20% 20% 20%. helicase 2. topoisomerases 3. DN polymerases 4. primase 5. DN ligase 20% 20% 20% 20% 20% 2 3 4 5 2 3 4 5 opyright 2009 Pearson Education, Inc. opyright 2009 Pearson Education, Inc. Which enzyme adds the RN bases which starts the new s? Which enzyme joins the Okazaki fragments on the lagging?. helicase 2. topoisomerases 3. DN polymerases 4. primase 5. DN ligase 20% 20% 20% 20% 20%. helicase 2. topoisomerases 3. DN polymerases 4. primase 5. DN ligase 20% 20% 20% 20% 20% 2 3 4 5 2 3 4 5 opyright 2009 Pearson Education, Inc. opyright 2009 Pearson Education, Inc. 3 --5 would pair with. 3 --5 2. 3 --5 3. 5 --3 4. 5 --3 hromosome consists of a DN molecule packed together with proteins he bacterial chromosome is a double-ed, circular DN molecule associated with a small amount of protein Eukaryotic chromosomes have linear DN molecules associated with a large amount of protein In a bacterium, the DN is supercoiled and found in a region of the cell called the nucleoid 7
5 m hromosome consists of a DN molecule packed together with proteins hromatin, a complex of DN and protein, is found in the nucleus of eukaryotic cells hromosomes fit into the nucleus through an elaborate, multilevel system of packing nimation: DN Packing Right-click slide / select Play Figure 6.22a Figure 6.22b hromatid (700 nm) 30-nm fiber Nucleosome (0 nm in diameter) DN double helix (2 nm in diameter) Loops Scaffold DN, the double helix Histones Histones Histone tail H Nucleosomes, or beads on a string (0-nm fiber) 30-nm fiber Looped domains (300-nm fiber) 300-nm fiber Replicated chromosome (,400 nm) Metaphase chromosome hromosome consists of a DN molecule packed together with proteins Figure 6.23 hromatin undergoes changes in packing during the cell cycle t interphase, some chromatin is organized into a 0-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping hough interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus 8
hromosome consists of a DN molecule packed together with proteins Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis DN wrapping around proteins Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Important oncepts Know the vocabulary in this lecture Structure of DN and their nucleotides he four bases, and which are paired together Be able to recognize the four base structures Know which bases are purines and Pyrimidines ype of bonds/linkages Be able to draw DN for me (you can use S and P for sugar and phosphate, for bases, 5 and 3 ) Important oncepts Be able to describe how is DN replicated Semiconservative replication Steps omplementary pairing Direction of building the complementary pair he role of helicase, Single- binding proteins opoisomerases, DN polymerases, DN ligase Understand how the leading is build vs how the lagging is built, know what Okazaki fragments are, Important oncepts Know what telomers and telomerases are What supplies the energy to be used to build the new Be able to identify correctly paired bases and incorrectly paired bases Know the repair mechanisms for DN 9