DNA Replication PDF

Topic 9: DNA Replication © 2017 Cengage Learning. All Rights Reserved. Digital Model of DNA © 2017 Cengage Learning. All Rights Reserved. Why It Matters... In 1868 Johann Miescher discovered “nuclein” (an acidic substance with a high phos...

Topic 9: DNA Replication © 2017 Cengage Learning. All Rights Reserved. Digital Model of DNA © 2017 Cengage Learning. All Rights Reserved. Why It Matters... In 1868 Johann Miescher discovered “nuclein” (an acidic substance with a high phosphorus content) in the nuclei of white blood cells It took more than 80 years to confirm that this material, now called DNA, was the molecule of inheritance Deoxyribonucleic acid (DNA) Molecule that is the genetic material of all living organisms © 2017 Cengage Learning. All Rights Reserved. Discovery of DNA Structure James Watson and Francis Crick’s discovery of the structure of the DNA molecule explained how genetic information is stored and replicated A molecular revolution was launched within biology, making it possible to relate genetic traits of living organisms to a universal molecular code present in the DNA of every cell © 2017 Cengage Learning. All Rights Reserved. The Molecular Revolution Watson and Crick with their 1953 model for DNA structure, which revolutionized the biological sciences © 2017 Cengage Learning. All Rights Reserved. Establishing DNA as the Hereditary Molecule Many scientists once believed that proteins were the most likely hereditary molecules Several experiments showed that DNA, not protein, is the genetic material © 2017 Cengage Learning. All Rights Reserved. 14.2 DNA Structure DNA contains four different nucleotides, each consisting of: One five-carbon sugar (deoxyribose) One phosphate group One of four nitrogenous bases (A, G, T, C) Adenine (A) and guanine (G) are purines Built from a pair of fused carbon-nitrogen rings Thymine (T) and cytosine (C) are pyrimidines Built from a single carbon-nitrogen ring © 2017 Cengage Learning. All Rights Reserved. Chargaff’s Rules Erwin Chargaff discovered that nitrogenous bases in DNA occur in definite ratios The amount of purines equals the amount of pyrimidines: Amount of adenine equals amount of thymine Amount of guanine equals amount of cytosine Chargaff’s rule: A = T, and G = C © 2017 Cengage Learning. All Rights Reserved. The Polynucleotide Chain DNA nucleotides are joined to form a polynucleotide chain Deoxyribose sugars are linked by phosphate groups in an alternating pattern, forming a sugar- phosphate backbone Each phosphate group links the 3′ carbon of one sugar with the 5′ carbon of the next sugar The entire linkage is a phosphodiester bond © 2017 Cengage Learning. All Rights Reserved. Polarity The polynucleotide chain of DNA has polarity At one end, a phosphate group is bound to the 5′ carbon of a deoxyribose sugar (the 5′ end) At the other end, a hydroxyl group is bound to the 3′ carbon of a deoxyribose sugar (the 3′ end) © 2017 Cengage Learning. All Rights Reserved. Four Subunits of DNA 5' end Phosphodiester bonds Phosphate connect adjacent Deoxyribose (a 5-carbon sugar) Adenine (A) Deoxyribonucleo deoxyribose sugars of tide the four subunits of Phosphodiest er bond Purines (double- DNA Guanine (G) ring structures) The polynucleotide chain has polarity Thymine (T) Pyrimidines (single-ring structures) Cytosine (C) Hydroxyl 3' group © 2017 end Cengage Learning. All Rights Reserved. Molecular Structure Maurice Wilkins and Rosalind Franklin, of King’s College, London, each used X-ray diffraction to study DNA structure Franklin interpreted an X-shaped distribution of spots in the diffraction pattern to mean that DNA has a helical structure X-ray diffraction An X-ray beam is directed at a molecule in the form of a regular solid (ideally a crystal) Positions of atoms in the molecule are deduced from diffraction patterns produced on photographic film © 2017 Cengage Learning. All Rights Reserved. X-Ray Diffraction of DNA A. X-ray diffraction analysis B. Franklin’s DNA diffraction of DNA pattern X-ray DNA source sample Science Source Beam of X- Photographic rays plate © 2017 Cengage Learning. All Rights Reserved. The DNA Model Watson and Crick constructed a double-helix model for DNA Two polynucleotide chains twist around each other, like a double-spiral staircase The two chains are antiparallel (opposite polarity) Pairs of bases fill the central space This arrangement satisfied both Franklin’s X-ray data and Chargaff‘s chemical analysis © 2017 Cengage Learning. All Rights Reserved. DNA Double Helix A. DNA double-helical structure B. Chemical C. Space-filling structure model overlaid 3' end with sugar– 2 5' end 3 phosphate nm 5' ' 5' end 3' end backbones 5-carbon sugar 5' end 3' (deoxyribos end e) Phospha te 10 base pairs group for each full twist of the Major DNA double helix = Nitrogenous groov 3.4 nm base e (guanine) Minor Distance between groov each e pair of bases = 0.34 nm 3' end 5' end 3 3' end 5' end ' 3' end 5' 5' end © 2017 Cengage Learning. All Rights Reserved. Base Pairs A purine and a pyrimidine, paired together, exactly fill the space between the two chains in the double helix The purine-pyrimidine base pairs in Watson and Crick’s model (A-T and G-C) are stabilized by hydrogen bonds – two between A and T and three between G and C C cannot pair with A, and G cannot pair with T, because of the hydrogen bonding requirements © 2017 Cengage Learning. All Rights Reserved. Complementary Strands The two strands of a DNA molecule are complementary to each other Complementary base pairing Wherever an A occurs in one strand, a T must be opposite it in the other strand Wherever a G occurs in one strand, a C must be opposite © 2017 Cengage Learning. All Rights Reserved. Hereditary Material Watson and Crick recognized that genetic information is coded into DNA by the linear sequence of the four nucleotides Combining the nucleotides into groups allows an essentially infinite number of different sequences to be “written” Watson, Crick, and Wilkins shared a Nobel Prize for their discovery in 1962 – Franklin died of cancer at age 38 in 1958 © 2017 Cengage Learning. All Rights Reserved. Study Break 14.2 1. Which bases in DNA are purines? Which are pyrimidines? 2. What bonds form between complementary base pairs? Between a base and the deoxyribose sugar? 3. Which features of the DNA molecule did Watson and Crick describe? 4. The percentage of A in a double-stranded DNA molecule is 20. What is the percentage of C in that DNA molecule? © 2017 Cengage Learning. All Rights Reserved. 14.3 DNA Replication Complementary base-pairing explains DNA replication Semiconservative replication Hydrogen bonds between the two strands break The two strands unwind and separate Each strand acts as a template for synthesis of a new, complementary strand Each new double helix has one old strand, derived from the parental DNA molecule, and one new strand © 2017 Cengage Learning. All Rights Reserved. Semiconservative Replication Watson and Crick’s model for DNA replication Complementar y base pairing in the DNA double helix: A Product molecules pairs with T, G pairs with C. are half old and half new Direction of replication The two chains unwind and separate. Each “old” strand is a template for the addition of bases according to the base- pairing rules. The result is two DNA Old helices that are exact copies of the parental Ne DNA molecule w with one “old” strand and one “new” strand. © 2017 Cengage Learning. All Rights Reserved. Other Proposed Models Conservative replication model The two strands of the original molecule serve as templates for the two strands of a new DNA molecule, then rewind into an all “old” molecule Dispersive replication model Neither parental strand is conserved and both chains of each replicated molecule contain old and new segments © 2017 Cengage Learning. All Rights Reserved. DNA Replication Models A. Semiconservative B. Conservative C. Dispersive replication replication replication 1st replicati on 2nd replicati on © 2017 Cengage Learning. All Rights Reserved. DNA Polymerases DNA polymerases assemble complementary polynucleotide chains from individual deoxyribonucleotides Four different deoxyribonucleoside triphosphates (one for each DNA base) are used: dATP, dGTP, dCTP, and dTTP DNA polymerase adds a nucleotide only to the 3′ end (the exposed hydroxyl group) of an existing nucleotide chain © 2017 Cengage Learning. All Rights Reserved. Antiparallel Strands A 3′–OH group is exposed at the “newest” end of a new DNA strand; the “oldest” end has an exposed 5′ triphosphate DNA polymerases only assemble nucleotide chains in the 5′→3′ direction Because DNA strands run antiparallel to each other, the template strand is “read” in the 3′→5′ direction © 2017 Cengage Learning. All Rights Reserved. 3 ' end New strand Template strand 3′ end 3′ end 5′ end 5′ end DNA polymerase 3′ end Pyrophosphate Incoming nucleotide 3′ end Deoxyribonucleoside triphosphate (dATP) Hydrolysis provides energy for DNA chain elongation Direction of reaction new chain 2 1 growth Stepped Art 5′ end 5′ end DNA Polymerase Structure DNA polymerases consist of several polypeptide subunits arranged in different “hand-shaped” domains Template DNA lies over the “palm” in a groove formed by the “fingers” and “thumb” The template strand and the 3′–OH of the new strand meet at the active site for the polymerization reaction of DNA synthesis, located in the palm domain © 2017 Cengage Learning. All Rights Reserved. DNA Polymerase Polymerization Templat reaction active site e base Pal m 5 Templat ' e strand 3 ' 5 OH ' 3' New P P P DNA strand Incoming Thum Finger b nucleotid s e © 2017 Cengage Learning. All Rights Reserved. Sliding DNA Clamp DNA polymerase extends the new DNA strand, one nucleotide at a time, by moving along the template Sliding DNA clamp Protein that encircles DNA and attaches to the rear of DNA polymerase (relative to forward movement) Tethers DNA polymerase to the template strand and increases rate of DNA synthesis © 2017 Cengage Learning. All Rights Reserved. DNA Polymerase and Sliding Clamp Template Sliding DNA polymerase strand clamp 3' 5' 5' 3 ' New DNA Direction of strand DNA synthesis © 2017 Cengage Learning. All Rights Reserved. Molecular Insights: Sliding Clamp Research Question: How is the sliding clamp loaded and unloaded onto replicating DNA in humans? Conclusion: The efficient unloading of sliding clamps by clamp loaders once DNA polymerase has dissociated from DNA is probably important for the overall efficiency of DNA replication © 2017 Cengage Learning. All Rights Reserved. Summary: DNA Replication The two strands of the DNA molecule unwind DNA polymerase adds nucleotides to an existing chain Direction of new synthesis is in the 5′→3′ direction, which is antiparallel to the template strand Nucleotides enter a newly synthesized chain according to the A-T and G-C complementary base-pairing rules © 2017 Cengage Learning. All Rights Reserved. Unwinding and Stabilizing DNA In the bacterial chromosome, unwinding of DNA for replication occurs at a small, specific region (origin of replication, ori ) DNA helicase unwinds the DNA strands, producing a Y-shaped replication fork Single-stranded binding proteins (SSBs) coat the exposed single-stranded DNA segments, keeping them from pairing Topoisomerase cuts and rejoins DNA to prevent twisting in circular bacterial chromosomes © 2017 Cengage Learning. All Rights Reserved. Helicase, SSBs, and Topoisomerase Origin of replication (ori) determines the start point for replication. 3 Replication ' fork 5 ' 3 5 Direction of ' unwinding ' DNA helicase, recruited by proteins that bind to the ori, uses energy of ATP hydrolysis to unwind the DNA Topoisomeras strands, producing the replication fork. e prevents 3 twisting as ' DNA unwinds. 5 ' 3 5 Direction of ' unwinding ' Single-stranded binding proteins (SSBs) coat and stabilize single- stranded DNA, preventing the two strands from reforming double-stranded DNA. © 2017 Cengage Learning. All Rights Reserved. RNA Primers and Primase DNA polymerases add nucleotides only to an existing strand A new strand begins with a short chain of RNA (primer), synthesized by the enzyme primase Primase leaves the template, and DNA polymerase takes over, extending the RNA primer with DNA nucleotides as it synthesizes the new DNA chain RNA primers are replaced with DNA later in replication © 2017 Cengage Learning. All Rights Reserved. RNA Primers and Primase (cont’d.) Primase synthesizes a short RNA primer to initiate a new DNA strand. 3' 5 5' ' 3' RNA primer Primase leaves; DNA polymerase takes over. 3' 5 5' ' 3' New DNA extended from DNA polymerase primer by DNA polymerase. © 2017 Cengage Learning. All Rights Reserved. Continuous and Discontinuous DNA Synthesis Because the two strands of a DNA molecule are antiparallel, only one template strand runs in a direction that allows DNA polymerase to make a continuous 5′→3′ copy DNA polymerase copies the other strand in short lengths (Okazaki fragments) synthesized in the direction opposite to that of DNA unwinding (discontinuous replication) © 2017 Cengage Learning. All Rights Reserved. Leading and Lagging Strands Leading strand In DNA replication, the new DNA strand is synthesized in the direction of DNA unwinding Synthesized on the leading strand template Lagging strand New DNA strand synthesized discontinuously, in the direction opposite DNA unwinding Synthesized on the lagging strand template © 2017 Cengage Learning. All Rights Reserved. Replication at a Replication Fork Leading strand template 3 ' Replication 3' Continuous fork 5 synthesis 5' ' Discontinuo 5 3' us 5' 5 3 ' ' ' synthesis 3' 3' 5 Direction of DNA unwinding ' Lagging strand template 3 ' 5 Leading 3 5' strand ' ' 3' 3 Lagging 5' ' strand 5 ' © 2017 Cengage Learning. All Rights Reserved. Enzymes in DNA Replication Many enzymes coordinate to replicate DNA DNA helicase unwinds DNA Primase initiates all new strands DNA polymerase III is the main polymerase DNA polymerase I forms the lagging strand DNA ligase binds Okazaki fragments together © 2017 Cengage Learning. All Rights Reserved.  Single-stranded binding proteins (SSBs) 3′ Replication fork Topoisomerase 5′ Primase 5′ RNA primers 5′ 3′ 3′ 5′ Direction of replication DNA helicase 1 (unwinding enzyme) DNA polymerase III Leading strand 3′ 5′ 3′ 5′ RNA DNA RNA 5′ 3′ 3′ 5′ Lagging strand 2 DNA polymerase III  DNA polymerase III 3′ 5′ DNA polymerase III 3′ 5′ 5′ 3′ 3′ 5′ 3′ 5′ 3 First Primer being Second Newly Okazaki extended by Okazaki synthesized fragment DNA polymerase III fragment primer DNA polymerase III 3′ 5′ 3′ 5′ Nick 5′ 3′ 3′ 5′ 4 3′ 5 5′ 3′ 5′ DNA ligase 5′ 3′ 3′ 5′ DNA polymerase III 3′ 5′ 6 Leading strand DNA polymerase III 3′ 5′ Lagging strand 5′ 3′ 3′ 5′ 3′ 5′ Newly Primer being synthesized extended by DNA primer polymerase Replication Bubbles and Multiple Origins Unwinding at an ori produces two replication forks, joined together to form a replication bubble Eukaryotic chromosomes have multiple origins – replication initiates and a replication bubble forms at each origin Forks eventually meet along the chromosomes to produce fully replicated DNA molecules © 2017 Cengage Learning. All Rights Reserved. Replication Bubble Replication bubble Origin of replication(ori) 3'5' 3' Lagging 5' Leading 3 5 5' strand strand 3' ' 3' ' 5 5 Leading Lagging ' 3 ' strand 5' strand Replication Replication ' 3' 5' 3' fork fork movement movement © 2017 Cengage Learning. All Rights Reserved. Multiple Origins of Replication Origi DNA double n helix Replication Replication forks direction © 2017 Cengage Learning. All Rights Reserved. Telomeres The RNA primer in DNA replication causes a problem for replicating linear chromosomes of eukaryotes When the primer is removed, it leaves a gap at the 5′ end of the new DNA strand that DNA polymerase can’t fill – causing the chromosome to shorten with each replication The ends of most eukaryotic chromosomes are protected by a buffer of noncoding DNA (the telomere) consisting of short, repeating sequences (the telomere repeat) © 2017 Cengage Learning. All Rights Reserved. Telomerase With each replication, some telomere repeats are lost, but the genes are unaffected Buffering fails when the entire telomere is lost Telomerase stops the shortening of telomeres by adding telomere repeats to chromosome ends An RNA section binds to DNA and is the template for addition of telomere repeats Active only in rapidly dividing embryonic cells, in germ cells – and in cancerous somatic cells © 2017 Cengage Learning. All Rights Reserved. Telomere Repeat Telomere repeats 5 3 1 End of a ' ' chromosome showing the primer 3 RNA 5 used for new DNA ' primer ' synthesis (red) still in place. Single-stranded region left after primer removal 5 3 2 Chromosome ' ' end after primer 5 removal. 3 ' ' Telomera se 3 Telomerase binds to the single- 5 3 stranded 3' end of ' ' the chromosome by 5 3 ' complementary base 3' pairing between the ' RNA 5' RNA of telomerase of and the telomere RNA template for telo- repeat. new telomere meras repeat DNA New e DNA 5 3 4 Telomerase ' ' synthe- sizes new 5 telomere DNA using 3 ' telomerase RNA as ' 3 5 the template. ' ' Single-stranded 5 The longer region left after (top) strand is primer removal replicated by 5 3 primase and DNA ' ' polymerase, and 5 Longer 5' 3 ' end of then the primer is removed, leaving a ' chromosome new 5' end to the bottom strand of © 2017 Cengage Learning. All Rights Reserved. the chromosome. Study Break 14.3 1. What is the importance of complementary base pairing to DNA replication? 2. Why is a primer needed for DNA replication? How is the primer made? 3. DNA polymerase III and DNA polymerase I are used in DNA replication in E. coli. What are their roles? 4. Why are telomeres important? © 2017 Cengage Learning. All Rights Reserved. 14.4 Repair of Errors in DNA Errors made during replication (base-pair mismatches) are corrected in a proofreading mechanism by DNA polymerases After replication is complete, remaining base-pair mismatches are corrected by a DNA repair mechanism © 2017 Cengage Learning. All Rights Reserved. Proofreading Mechanism The proofreading mechanism allows DNA polymerases to back up and remove mispaired nucleotides If a newly added nucleotide is mismatched, DNA polymerase reverses, using 3′→5′ exonuclease activity to remove the newly added incorrect nucleotide DNA polymerase then resumes forward synthesis and inserts the correct nucleotide © 2017 Cengage Learning. All Rights Reserved. Proofreading Templat DNA e polymerase strand Polymerization activity of DNA polymerase adds DNA nucleotides to the new chain in the 5' 3' direction using complementary base pairing rules. New strand Rarely, DNA polymerase adds a mispaired nucleotide. New strand DNA polymerase recognizes the mismatched base pair.The enzyme reverses, using its 3' 5' exonuclease to remove the mispaired nucleotide from the strand. DNA polymerase resumes its polymerization activity in the forward direction, extending the new chain in the 5' 3' direction. © 2017 Cengage Learning. All Rights Reserved. DNA Repair Mechanisms DNA repair mechanisms correct base-pair mismatches that remain after proofreading (mismatch repair) Distortions in DNA structure caused by mispaired bases provide recognition sites for mismatch repair enzymes Repair enzymes cut the new DNA strand on each side of the mismatch, and remove a portion of the chain DNA polymerase fills the gap with new DNA, and DNA ligase seals the nucleotide chain © 2017 Cengage Learning. All Rights Reserved. Mismatch Repair Base-pair mismatch Template strand Mismatch repair proteins scan DNA for a mispaired base that was missed in proofreading and cleave the backbone of the new strand on each side of the mismatch. New strand The repair proteins remove several to many bases, including the mismatched base, leaving a gap in the DNA. A repair DNA polymerase fills in the gap with its 5' 3' polymerizing activity, using the template strand as a guide. Nick left after gap filled in DNA ligase seals the nick left after gap filling to © 2017 complete Cengage Learning. the repair. All Rights Reserved. Excision Repair DNA damage occurs constantly in cells Base-excision repair mechanisms repair nonbulky damage by removing the erroneous base and replacing it with the correct one based on complementary pairing rules Nucleotide-excision repair repairs bulky distortions in DNA (such as in thymine dimers) by removing an entire segment of DNA © 2017 Cengage Learning. All Rights Reserved. Thymine Dimer Thymine dimer Bulky distortion of DNA © 2017 Cengage Learning. All Rights Reserved. Mutation and Evolutionary Processes Errors that remain in DNA after proofreading and DNA repair are a primary source of mutations (changes in DNA sequence that are passed on in replicated copies) Mutation in a gene can alter the protein encoded by the gene, which may alter how the organism functions – mutations are the ultimate source of variability acted on by natural selection © 2017 Cengage Learning. All Rights Reserved.