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Biochemistry Biochemistry • Ch1~Ch14: Introductionà Biochem 1 • Ch15~Ch27: Metabolism • Ch28~Ch32: Molecular Biology – Ch4 DNA • Ch33: Sensory Systems • Ch34: Immunology • Ch35: Molecular Motors • Ch36: Drug Development 1 Chapter 28 DNA Replication, Repair, and Recombination 2 Perhaps the most exciting aspect of the structure of DNA deduced by Watson and Crick was, as expressed in their words, that the “specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” A double helix separated into two single strands can be replicated because each strand serves as a template on which its complementary strand can be assembled (Figure 28.1). 3 To preserve the information encoded in DNA through many cell divisions, copying of the genetic information must be extremely faithful. To replicate the human genome without mistakes, an error rate of less than 1 bp per 3 × 109 bp must be achieved. Such remarkable accuracy is achieved through a multilayered system - Accurate DNA synthesis (an error rate of 1 per 103–104 bases) - Proofreading during DNA synthesis (which reduces that error rate to approximately 1 per 106–107 bp) - Postreplication mismatch repair (which reduces the error rate to approximately 1 per 109–1010 bp). 4 Even after DNA has been initially replicated, the genome is still not safe. Although DNA is remarkably robust, ultraviolet light as well as a range of chemical species can damage DNA, introducing changes in the DNA sequence (mutations) or lesions that can block further DNA replication (Figure 28.2). 5 All organisms contain DNA-repair systems that detect DNA damage and act to preserve the original sequence. Mutations in genes that encode components of DNA-repair systems are key factors in the development of cancer. Among the most potentially devastating types of DNA damage are double-stranded breaks in DNA. With both strands of the double helix broken in a local region, neither strand is intact to act as a template for future DNA synthesis 6 A mechanism used to repair such lesions relies on DNA Recombination—that is, the reassortment of DNA sequences present on two different double helices. In addition to its role in DNA repair, Recombination is crucial for the generation of genetic diversity in meiosis. Recombination is also the key to generating a highly diverse repertoire of genes for key molecules in the immune system (Chapter 34). 7 4.1 A Nucleic Acid Consists of Four Kinds of Bases Linked to a Sugar-Phosphate Backbone Linear polymers, Monomer: Nucleotide A single nucleotide unit consists of three components: a sugar, a phosphate, and one of four bases. 8 RNA and DNA Differ in the Sugar Component and One of the Bases The sugar in deoxyribonucleic acid (DNA) is deoxyribose. Note that sugar carbons are numbered with primes to differentiate them from atoms in the bases. The sugars in nucleic acids are linked to one another by phosphodiester bridges. The chain of sugars linked by phosphodiester bridges is referred to as the backbone of the nucleic acid Whereas the backbone is constant in a nucleic acid, the bases vary from one monomer to the next. 9 Two of the bases of DNA are derivatives of purine : adenine (A) and guanine (G) and two of pyrimidine : cytosine (C) and thymine (T ) The covalent structure of RNA differs from that of DNA in two respects. First, the sugar units in RNA are riboses rather than deoxyriboses. Ribose contains a 2' -hydroxyl group not present in deoxyribose. Second, one of the four major bases in RNA is uracil (U) instead of thymine (T). Why ribose and uracil? Why phosphate? 10 Nucleotides Are the Monomeric Units of Nucleic Acids • Nucleoside • Nucleotide = nucleoside tri-phosphate • Polarity: ACG vs. GCA By convention, the base sequence is written in the 5’-to-3’ direction 11 4.2 A Pair of Nucleic Acid Chains with Complementary Sequences Can Form a Double-Helical Structure Maurice Wilkins and Rosalind Franklin obtained x-ray diffraction photographs of fibers of DNA. The characteristics of these diffraction patterns indicated that DNA is formed of two chains that wind in a regular helical structure. From these data and others, James Watson and Francis Crick deduced a structural model for DNA that accounted for the diffraction pattern and was the source of some remarkable insights into the functional properties of nucleic acids 12 13 The features of the Watson- Crick model of DNA 1. Two helical polynucleotide chains are coiled around a common axis with a right-handed screw sense. The chains are antiparallel, opposite polarity 2. The sugar–phosphate backbones are on the outside and the purine and pyrimidine bases lie on the inside of the helix. 3. The bases are nearly perpendicular to the helix axis, and adjacent bases are separated by 3.4 Å from diffraction pattern. The helical structure repeats every 34 Å, and so there are 10 bases per turn of helix. Each base is rotate 36 degrees (360/10). 4. The diameter of the helix is 20 Å. 14 How is such a regular structure able to accommodate an arbitrary sequence of bases, given the different sizes and shapes of the purines and pyrimidines? In attempting to answer this question, Watson and Crick discovered that guanine can be paired with cytosine and adenine with thymine to form base pairs that have essentially the same shape. These base pairs are held together by specific hydrogen bonds, which, although weak (4–21 kJ mol-l ), stabilize the helix because of their large numbers in a DNA molecule. These base-pairing rules account for the observation, originally made by Erwin Chargaff in 1950 15 4.4 DNA Is Replicated by Polymerases That Take Instructions from Templates We now turn to the molecular mechanism of DNA replication. The full replication machinery in a cell comprises more than 20 proteins engaged in intricate and coordinated interplay. In 1958, Arthur Kornberg and his colleagues isolated from E. coli the first known of the enzymes, called DNA polymerases, that promote the formation of the bonds joining units of the DNA backbone. Arthur Kornberg Roger Kornberg Isolated DNA polymerase I Studied eukaryotic Nobel Prize in 195' transcription process of RNA polymerase Nobel Prize in 2006 16 DNA Polymerase Catalyzes phosphodiester-bridge Formation DNA polymerases catalyze the step-by-step addition of deoxyribonucleotide units to a DNA chain DNA synthesis has the following characteristics: 1. The reaction requires all four activated precursors dNTP: dATP, dGTP, dCTP, TTP & Mg2+ 2. The new DNA chain is assembled directly on a preexisting DNA template. DNA polymerase is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template. 17 3. DNA polymerases require a primer to begin synthesis. A primer strand having a free 3'-OH group The chain-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3' -OH terminus of the growing chain on the innermost phosphorus atom of the deoxynucleoside triphosphate. A phosphodiester bridge is formed and pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase helps drive the polymerization forward. Elongation of the DNA chain proceeds in the 5' -to-3' direction. 18 4. Many DNA polymerases are able to correct mistakes in DNA by removing mismatched nucleotides. These polymerases have a distinct nuclease activity that allows them to excise incorrect bases by a separate reaction. This nuclease activity contributes to the remarkably high fidelity of DNA replication, which has an error rate of less than 10-8 per base pair. 19 28.1 DNA Replication Proceeds by the Polymerization of Deoxyribonucleoside Triphosphates Along a Template • Each strand within the parent double helix acts as a template for the synthesis of a new DNA strand with a complementary sequence. • The building blocks for the synthesis of the new strands are deoxyribonucleoside triphosphates. • They are added, one at a time, to the 3’ end of an existing strand of DNA. 20 Although this reaction is in principle quite simple, it is significantly complicated by specific features of the DNA double helix. First, the two strands of the double helix run in opposite directions. 5’-to-3’ direction Second, the two strands must be separated Finally, the two strands of the double helix wrap around each other. Thus, strand separation also entails the unwinding of the double helix. This unwinding creates supercoils that must themselves be resolved as replication continues. 21 DNA polymerases require a template and a primer DNA polymerases add nucleotides to the 3’ end of a polynucleotide chain. (see Figure 4.25). 22 All DNA polymerases have structural features in common The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure was elucidated by Tom Steitz and coworkers, who determined the structure of the so-called Klenow fragment of DNA polymerase I from E. coli (Figure 28.3). This approximates the shape of a right hand with domains that are referred to as the fingers, the thumb, and the palm. In addition to the polymerase, the Klenow fragment includes a domain with 3’ à 5’ exonuclease activity that participates in proofreading and correcting the polynucleotide product. 23 24 DNA polymerases are remarkably similar in overall shape, although they differ substantially in detail. At least five structural classes have been identified; some of them are clearly homologous, whereas others appear to be the products of convergent evolution. In all cases, the finger and thumb domains wrap around DNA and hold it across the enzyme’s active site, which comprises residues primarily from the palm domain. Furthermore, all DNA polymerases use similar strategies to catalyze the polymerase reaction, making use of a mechanism in which two metal ions take part.
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  • Klenow Fragment, #EP0054
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