BCH 5045 Graduate Survey of Biochemistry...Images from the Text are protected by Copyright (c) 2008...

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BCH 5045

Graduate Survey of Biochemistry

Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham

Lecture 18

Slide sets available at: http://hort.ifas.ufl.edu/teach/guyweb/bch5045/index.html

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• LEHNINGER • PRINCIPLES OF BIOCHEMISTRY

• Fifth Edition

David L. Nelson and Michael M. Cox

© 2008 W. H. Freeman and Company

CHAPTER 8 Nucleotides and Nucleic Acids



Bad things can happen to DNA. Chemically or physicochemically the bases of DNA can be modified and when this happens can cause mutations. Some examples are shown here.

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Presentation Notes

FIGURE 8-30a Some well-characterized nonenzymatic reactions of nucleotides. (a) Deamination reactions. Only the base is shown. FIGURE 8-30b Some well-characterized nonenzymatic reactions of nucleotides. (b) Depurination, in which a purine is lost by hydrolysis of the N-β-glycosyl bond. Loss of pyrimidines via a similar reaction occurs, but much more slowly. The resulting lesion, in which the deoxyribose is present but the base is not, is called an abasic site or an AP site (apurinic site or, rarely, apyrimidinic site). The deoxyribose remaining after depurination is readily converted from the β-furanose to the aldehyde form (see Figure 8-3). Further nonenzymatic reactions are illustrated in Figures 8-31 and 8-32



To Paraphrase Willie Nelson: Mamas, don't let your babies grow up to be sunbathers. UV light, can modify DNA causing mutations by the dimerization of adjacent thymines to form a cyclobutane ring or a 6-4 photoproduct as shown on right.

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Presentation Notes

FIGURE 8-31a Formation of pyrimidine dimers induced by UV light. (a) One type of reaction (on the left) results in the formation of a cyclobutyl ring involving C-5 and C-6 of adjacent pyrimidine residues. An alternative reaction (on the right) results in a 6-4 photoproduct, with a linkage between C-6 of one pyrimidine and C-4 of its neighbor. FIGURE 8-31b Formation of pyrimidine dimers induced by UV light. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA



What does it mean alkylating agent? Any of several highly reactive chemical compounds that bond with various nucleophilic groups in nucleic acids and proteins and cause mutagenic, carcinogenic, or cytotoxic effects. Methylation is the most common type of alkylation.

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FIGURE 8-32a Chemical agents that cause DNA damage. (a) Precursors of nitrous acid, which promotes deamination reactions.



DNA sequencing and DNA synthesis

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FIGURE 8-33a DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3′-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond.



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FIGURE 8-36 Nucleoside phosphates. General structure of the nucleoside 5′-mono-, di-, and triphosphates (NMPs, NDPs, and NTPs) and their standard abbreviations. In the deoxyribonucleoside phosphates (dNMPs, dNDPs, and dNTPs), the pentose is 2′-deoxy-D-ribose.



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FIGURE 8-37 The phosphate ester and phosphoanhydride bonds of ATP. Hydrolysis of an anhydride bond yields more energy than hydrolysis of the ester. A carboxylic acid anhydride and carboxylic acid ester are shown for comparison.



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FIGURE 8-38 (part 1) Some coenzymes containing adenosine. The adenosine portion is shaded in light red. Coenzyme A (CoA) functions in acyl group transfer reactions; the acyl group (such as the acetyl or acetoacetyl group) is attached to the CoA through a thioester linkage to the β-mercaptoethylamine moiety. NAD+ functions in hydride transfers, and FAD, the active form of vitamin B2 (riboflavin), in electron transfers. Another coenzyme incorporating adenosine is 5′-deoxyadenosylcobalamin, the active form of vitamin B12 (see Box 17-2), which participates in intramolecular group transfers between adjacent carbons.



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FIGURE 8-38 (part 2) Some coenzymes containing adenosine. The adenosine portion is shaded in light red. Coenzyme A (CoA) functions in acyl group transfer reactions; the acyl group (such as the acetyl or acetoacetyl group) is attached to the CoA through a thioester linkage to the β-mercaptoethylamine moiety. NAD+ functions in hydride transfers, and FAD, the active form of vitamin B2 (riboflavin), in electron transfers. Another coenzyme incorporating adenosine is 5′-deoxyadenosylcobalamin, the active form of vitamin B12 (see Box 17-2), which participates in intramolecular group transfers between adjacent carbons. FIGURE 8-38 (part 3) Some coenzymes containing adenosine. The adenosine portion is shaded in light red. Coenzyme A (CoA) functions in acyl group transfer reactions; the acyl group (such as the acetyl or acetoacetyl group) is attached to the CoA through a thioester linkage to the β-mercaptoethylamine moiety. NAD+ functions in hydride transfers, and FAD, the active form of vitamin B2 (riboflavin), in electron transfers. Another coenzyme incorporating adenosine is 5′-deoxyadenosylcobalamin, the active form of vitamin B12 (see Box 17-2), which participates in intramolecular group transfers between adjacent carbons.



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FIGURE 8-39 Three regulatory nucleotides.



How are nucleic acid strands (RNA or DNA) synthesized? It is way easier than you think. What functional groups are required for DNA or RNA polymerization?

The chemistry of the nucleotide triphosphates which derived from the chemistry of the phosphate group are key, and the hydroxyl group at the 3 position of the deoxyribose, or ribose also for that matter. Also the free energy available in the phosphate anhydride bonds upon hydrolysis is central to formation of the phosphodiester linkages in the formation of nucleic acids.

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FIGURE 8-33a DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3′-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond.



This analog was the stroke of genius by Sanger in developing the most widely used method of DNA sequencing. What does the dd mean?

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FIGURE 8-33b DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (b) The Sanger sequencing procedure uses dideoxynucleoside triphosphate (ddNTP) analogs to interrupt DNA synthesis. (The Sanger method is also known as the dideoxy method.) When a ddNTP is inserted in place of a dNTP, strand elongation is halted after the analog is added, because it lacks the 3′-hydroxyl group needed for the next step.



Here the Sanger dideoxy method of DNA sequencing is illustrated. The Key elements are primers, dideoxy nucleotide triphosphates, regular deoxynucleotide triphosphates attached to fluorescent tags, template DNA and a DNA polymerase. Can you tell how this works?

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FIGURE 8-33c DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (c) The DNA to be sequenced is used as the template strand, and a short primer, radioactively or fluorescently labeled, is annealed to it. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some locations where dC normally occurs. Given the excess of dCTP over ddCTP, the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high probability of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each ending with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the different-sized fragments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5′ end), and the sequence is read (in the 5′→3′ direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed.

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