Nucleic Acids PDF

Nucleic Acids 22 CHAPTER O UT LI N E...

Nucleic Acids 22 CHAPTER O UT LI N E 22.1 Types of Nucleic Acids 798 22.2 Nucleotide Building Blocks 799 22.3 Nucleotide Formation 800 22.4 Primary Nucleic Acid Structure 802 Chemistry at a Glance Nucleic Acid Structure 805 22.5 The DNA Double Helix 806 22.6 Replication of DNA Molecules 809 Dr. Nikos/James Burns/Phototake Chemistry at a Glance DNA Replication 812 22.7 Overview of Protein Synthesis 814 22.8 Ribonucleic Acids 814 22.9 Transcription: RNA Synthesis 815 22.10 The Genetic Code 819 Human egg and sperm. 22.11 Anticodons and tRNA Molecules 822 22.12 Translation: Protein Synthesis 825 Chemistry at a Glance A most remarkable property of living cells is their ability to produce exact Protein Synthesis: Transcription replicas of themselves. Furthermore, cells contain all the instructions and Translation 829 needed for making the complete organism of which they are a part. The molecules within a cell that are responsible for these amazing capabilities are 22.13 Mutations 830 nucleic acids. 22.14 Nucleic Acids and The Swiss physiologist Friedrich Miescher (1844–1895) discovered nucleic Viruses 833 acids in 1869 while studying the nuclei of white blood cells. The fact that they 22.15 Recombinant DNA and were initially found in cell nuclei and are acidic accounts for the name nucleic Genetic Engineering 834 acid. Although it is now known that nucleic acids are found throughout a cell, 22.16 The Polymerase Chain not just in the nucleus, the name is still used for such materials. Reaction 838 Chemical Connections 22-A Antimetabolites: Anticancer 22.1 Types of Nucleic Acids Drugs That Inhibit DNA Synthesis 813 Two types of nucleic acids are found within cells of higher organisms: deoxyri- 22-B Antibiotic Protein Synthesis bonucleic acid (DNA) and ribonucleic acid (RNA). Nearly all the DNA is found Inhibitors 831 within the cell nucleus. Its primary function is the storage and transfer of genetic information. This information is used (indirectly) to control many functions of a living cell. In addition, DNA is passed from existing cells to new cells during cell division. RNA occurs in all parts of a cell. It functions primarily in synthesis of Sign in to OWL at www.cengage.com/owl to view tutorials and simulations, develop proteins, the molecules that carry out essential cellular functions. The structural problem-solving skills, and complete online distinctions between DNA and RNA molecules are considered in Section 22.4. homework assigned by your professor. 798  22.2 Nucleotide Building Blocks 799 All nucleic acid molecules are unbranched polymers. A nucleic acid is an It was not until 1944, 75 years unbranched polymer in which the monomer units are nucleotides. Thus the starting after the discovery of nucleic acids, that scientists obtained the first point for a discussion of nucleic acids is an understanding of the structures and evidence that these molecules are chemical properties of nucleotides. responsible for the storage and transfer of genetic information. 22.2 Nucleotide Building Blocks A nucleotide is a three-subunit molecule in which a pentose sugar is bonded to both a Proteins are polypeptides, many car- phosphate group and a nitrogen-containing heterocyclic base. With a three-subunit bohydrates are polysaccharides, and structure, nucleotides are more complex monomers than the monosaccharides nucleic acids are polynucleotides. of polysaccharides (Section 18.8) and the amino acids of proteins (Section 20.2). A block structural diagram for a nucleotide is Base Phosphate Sugar Pentose Sugars The sugar unit of a nucleotide is either the pentose ribose or the pentose 29-deoxyribose. The systems for numbering the atoms in the pentose and nitrogen- 59 59 HOCH2 O HOCH2 O containing base subunits of a nucle- OH OH otide are important and will be used 49 19 49 19 extensively in later sections of this 39 29 39 29 chapter. The convention is that OH OH OH H 1. Pentose ring atoms are desig- b -D-Ribose b -D-29-Deoxyribose nated with primed numbers. 2. Nitrogen-containing base ring Structurally, the only difference between these two sugars occurs at carbon 29. atoms are designated with The 9OH group present on this carbon in ribose becomes a 9H atom in unprimed numbers. 29-deoxyribose. (The prefix deoxy- means “without oxygen.”) RNA and DNA differ in the identity of the sugar unit in their nucleotides. In RNA, the sugar unit is ribose—hence the R in RNA. In DNA, the sugar unit is 29-deoxyribose—hence the D in DNA. Nitrogen-Containing Heterocyclic Bases Five nitrogen-containing heterocyclic bases are nucleotide components. Three of Pyrimidine and purine do not them- them are derivatives of pyrimidine (Section 17.9), a monocyclic base with a six- selves occur naturally; numerous derivatives of these two com- membered ring, and two are derivatives of purine (Section 17.9), a bicyclic base pounds, however, are naturally with fused five- and six-membered rings. occurring substances. 4 7 6 3 N 5 1 5 N N 8 A pyrimidine derivative that was 6 2 9 2 N1 N 4 N encountered previously is the B vita- A 3 min thiamine (see Section 21.14). H Pyrimidine Purine Caffeine, the most widely used Both of these heterocyclic compounds are bases because they contain amine func- nonprescription central nervous tional groups (secondary or tertiary), and amine functional groups exhibit basic system stimulant, is the 1,3,7- behavior (proton acceptors; Section 17.6). trimethyl-2,6-dioxo derivative The three pyrimidine derivatives found in nucleotides are thymine (T), cyto- of purine (Section 17.9). sine (C), and uracil (U). O NH2 O H H CH3 4 E 4 4 E 5 N N N 2 2 2 N O N O N O A A A H H H Thymine (T) Cytosine (C) Uracil (U) Thymine is the 5-methyl-2,4-dioxo derivative, cytosine the 4-amino-2-oxo deriva- tive, and uracil the 2,4-dioxo derivative of pyrimidine. 800 Chapter 22 Nucleic Acids The two purine derivatives found in nucleotides are adenine (A) and guanine (G). NH2 O H N N 6 E 6 N N 2 N N N N NH2 f f H H Adenine (A) Guanine (G) Figure 22.1 Space-filling model Adenine is the 6-amino derivative of purine, and guanine is the 2-amino-6-oxo of the molecule adenine, a purine derivative. A space-filling model for adenine is shown in Figure 22.1. nitrogen-containing heterocyclic Adenine, guanine, and cytosine are found in both DNA and RNA. Uracil base present in both DNA and is found only in RNA, and thymine usually occurs only in DNA. Figure 22.2 RNA. summarizes the occurrences of nitrogen-containing heterocyclic bases in nucleic acids. Phosphate Phosphate, the third component of a nucleotide, is derived from phosphoric acid (H3PO4). Under cellular pH conditions, the phosphoric acid loses two of its hydro- gen atoms to give a hydrogen phosphate ion (HPO422). OH O2 A A O P P O OH O P P O OH 1 2H 1 A A OH O2 Phosphoric acid Hydrogen phosphate ion 22.3 Nucleotide Formation The formation of a nucleotide from a sugar, a base, and a phosphate can be visual- ized as a two-step process. Nucleoside 5 Sugar 1 Base 1. First, the pentose sugar and nitrogen-containing base react to form a two- Nucleotide 5 Nucleoside 1 subunit entity called a nucleoside (not nucleotide, s versus t). Phosphate 2. The nucleoside reacts with a phosphate group to form the three-subunit entity called a nucleotide. It is nucleotides that become the building blocks for nucleic acids. Figure 22.2 Two purine bases In both DNA and RNA and three pyrimidine bases are found in the nucleotides present NH2 O N H in nucleic acids. N N N N N N N N N N N NH2 H H H Purine Adenine Guanine A G O NH2 O CH3 H H To remember which two of the five N N N N nucleotide bases are the purine derivatives (fused rings), use the N O N O N O phrase “pure silver” and substitute N the chemical symbol for silver, Pyrimidine H H H which is Ag. Thymine Cytosine Uracil T C U pure Ag purine A and G In DNA In RNA  22.3 Nucleotide Formation 801 Nucleoside Formation A nucleoside is a two-subunit molecule in which a pentose sugar is bonded to a nitrogen- containing heterocyclic base. The following structural equation is representative of nucleoside formation. Base O B H N 7 NE O B 8 N H 9 HNH 7 NE N N 2 8 59 H2O HOOOCH2 O 9 HNH H N N 2 49 19 59 HOOOCH2 O 39 29 OH 1 H2O 49 19 OH OH 39 29 OH OH Sugar Nucleoside Important characteristics of the nucleoside formation process of combining two molecules into one are: 1. The base is always attached to C-19 of the sugar (the anomeric carbon atom (Section 18.10)), which is always in a b-configuration. For purine bases, attachment is through N-9; for pyrimidine bases, N-1 is involved. The bond connecting the sugar and base is a b-N-glycosidic linkage (Section 18.3). 2. A molecule of water is formed as the two molecules bond together; a conden- sation reaction occurs. Eight nucleosides are associated with nucleic acid chemistry—four involve ribose (RNA nucleosides) and four involve deoxyribose (DNA nucleosides). The eight combinations are: RNA Nucleosides DNA Nucleosides ribose-adenine deoxyribose-adenine ribose-cytosine deoxyribose-cytosine ribose-guanine deoxyribose-guanine ribose-uracil deoxyribose-thymine Nucleosides are named as derivatives of the base that they contain; the base’s name is modified using a suffix. 1. For pyrimidine bases, the suffix -idine is used (cytidine, thymidine, uridine). 2. For purine bases, the suffix -osine is used (adenosine, guanosine). 3. The prefix -deoxy is used to indicate that the sugar present is deoxyribose. No prefix is used when the sugar present is ribose. Using these rules, the nucleoside containing ribose and adenine is called adenosine, and the nucleoside containing deoxyribose and thymine is called deoxythymidine. Nucleotide Formation Addition of a phosphate group to a nucleoside produces a nucleotide. The follow- ing structural equation is representative of nucleotide formation. O O B H B H N N 7 NE O2 7 NE H2O 8 A 59 8 O2 9 HNH OPPOOOCH2 O 9 HNH A N N 2 N N 2 59 A OPPOOH HOOOCH2 O O O2 49 19 A 39 29 O2 49 19 1 H2O 39 29 OH OH OH OH Phosphate Nucleoside Nucleotide 802 Chapter 22 Nucleic Acids Table 22.1 Information Concerning the Eight Nucleotides That Are Building Blocks for DNA and RNA Base Abbreviation Nucleoside Nucleotide Abbreviation DNA Adenine A Deoxyadenosine Deoxyadenosine 59-monophosphate dAMP Guanine G Deoxyguanosine Deoxguanosine 59-monophosphate dGMP Cytosine C Deoxycytidine Deoxycytidine 59-monophosphate dCMP Thymine T Deoxythymidine Deoxythymidine 59-monophosphate dTMP RNA Adenine A Adenosine Adenosine 59-monophosphate AMP Guanine G Guanosine Guanosine 59-monophosphate GMP Cytosine C Cytidine Cytidine 59-monophosphate CMP Uracil U Uridine Uridine 59-monophosphate UMP Important characteristics of the nucleotide formation process of adding a phosphate group to a nucleoside are the following: 1. The phosphate group is attached to the sugar at the C-59 position through a phosphate-ester linkage. 2. As with nucleoside formation, a molecule of water is produced in nucleotide formation. Thus, overall, two molecules of water are produced in combining a sugar, base, and phosphate into a nucleotide. Nucleotides are named by appending the term 59-monophosphate to the name of the nucleoside from which they are derived. Addition of a phosphate group to the nucleoside adenosine produces the nucleotide adenosine 59-monophosphate. Abbreviations for nucleotides exist, which are used in a manner similar to that for amino acids (Section 20.2). The abbreviations use the one-letter symbols for the base (A, C, G, T, and U ), MP for monophosphate, and a lowercase d at the start of the abbreviation when deoxyribose is the sugar. The abbreviation for adeno- sine 59-monophosphate is AMP and that for deoxyadenosine 59-monophosphate is dAMP. Table 22.1 summarizes information presented in this section about nucleo- sides and nucleotides. 22.4 Primary Nucleic Acid Structure Nucleotides are related to nucleic Nucleic acids are polymers in which the repeating units, the monomers, are nucleo- acids in the same way that amino tides (Section 22.2). The nucleotide units within a nucleic acid molecule are linked acids are related to proteins. to each other through sugar–phosphate bonds. The resulting molecular structure (Figure 22.3) involves a chain of alternating sugar and phosphate groups with a base group protruding from the chain at regular intervals. In Section 22.1, the two general types of nucleic acids—ribonucleic acids and deoxyribonucleic acids—were mentioned, but their definitions were not given. Defi- nitions are now in order. A ribonucleic acid (RNA) is a nucleotide polymer in which each of the monomers contains ribose, a phosphate group, and one of the heterocyclic bases adenine, cytosine, guanine, or uracil. Two changes to this definition generate the deoxyribonucleic acid definition; deoxyribose replaces ribose and thymine replaces uracil. A deoxyribonucleic acid (DNA) is a nucleotide polymer in which each Figure 22.3 The general structure of a nucleic acid in terms of Base Base Base nucleotide subunits. Phosphate Sugar Phosphate Sugar Phosphate Sugar Nucleotide Nucleotide Nucleotide  22.4 Primary Nucleic Acid Structure 803 Figure 22.4 (a) The generalized Phosphate Phosphate Phosphate backbone structure of a nucleic Sugar Deoxyribose Ribose acid. (b) The specific backbone structure for a deoxyribonucleic Phosphate Phosphate Phosphate acid (DNA). (c) The specific backbone structure for a Sugar Deoxyribose Ribose ribonucleic acid (RNA). Phosphate Phosphate Phosphate Sugar Deoxyribose Ribose a Nucleic Acid b DNA c RNA Backbone Backbone Backbone of the monomers contains deoxyribose, a phosphate group, and one of the heterocyclic bases adenine, cytosine, guanine, or thymine. The alternating sugar–phosphate chain in a nucleic acid structure is often called The backbone of a nucleic acid the nucleic acid backbone. This backbone is constant throughout the entire nucleic structure is always an alternating sequence of phosphate and sugar acid structure. For DNA molecules, the backbone consists of alternating phosphate groups. The sugar is ribose in RNA and deoxyribose sugar units; for RNA molecules, the backbone consists of alternat- and deoxyribose in DNA. ing phosphate and ribose sugar units. Figure 22.4 contrasts the generalized backbone structure for a nucleic acid with the specific backbone structures of DNAs and RNAs. The variable portion of nucleic acid structure is the sequence of bases attached to the sugar units of the backbone. The sequence of these base side chains distin- guishes various DNAs from each other and various RNAs from each other. Only four types of bases are found in any given nucleic acid structure. This situation is much simpler than that for proteins, where 20 side-chain entities (amino acids) are available (Section 20.2). In both RNA and DNA, adenine, guanine, and cytosine are encountered as side-chain components; thymine is found mainly in DNA, and uracil is found only in RNA (Figure 22.2). Primary nucleic acid structure is the sequence in which nucleotides are linked Just as the order of amino acid together in a nucleic acid. Because the sugar–phosphate backbone of a given nucleic side chains determines the primary acid does not vary, the primary structure of the nucleic acid depends only on the structure of a protein (Section 20.10), the order of nucleotide sequence of bases present. Further information about nucleic acid structure can bases determines the primary be obtained by considering the detailed four-nucleotide segment of a DNA mole- structure of a nucleic acid. cule shown in Figure 22.5. The following list describes some important points about nucleic acid struc- ture that are illustrated in Figure 22.5: 1. Each nonterminal phosphate group of the sugar–phosphate backbone is bonded to two sugar molecules through a 39,59-phosphodiester linkage. There is a phosphoester bond to the 59 carbon of one sugar unit and a phosphoester bond to the 39 carbon of the other sugar. 2. A nucleotide chain has directionality. One end of the nucleotide chain, the For both nucleic acids and proteins, 59 end, normally carries a free phosphate group attached to the 59 carbon a distinction is made between the two ends of the polymer chain. For atom. The other end of the nucleotide chain, the 39 end, normally has a free nucleic acids, there is a 59 end and a hydroxyl group attached to the 39 carbon atom. By convention, the sequence 39 end; for proteins, there is an of bases of a nucleic acid strand is read from the 59 end to the 39 end. N-terminal end and a C-terminal 3. Each nonterminal phosphate group in the backbone of a nucleic acid carries end (Section 20.7). a 21 charge. The parent phosphoric acid molecule from which the phosphate was derived originally had three 9OH groups (Section 22.2). Two of these become involved in the 39,59-phosphodiester linkage. The remaining 9OH group is free to exhibit acidic behavior—that is, to produce a H1 ion. O O B B O OO P OOO O OO P OOO 1 H1 A A OH O2 This behavior by the many phosphate groups in a nucleic acid backbone gives nucleic acids their acidic properties. 804 Chapter 22 Nucleic Acids Figure 22.5 A four-nucleotide- Thymine long segment of DNA. (The choice O of bases was arbitrary.) CH3 H 5' end N O– N O 5' O P O CH2 O O– 4' Guanine 2' O 3' H N H N O N N NH2 5' 3',5'-Phosphodiester O P O CH2 O linkage O– 4' Cytosine 2' NH2 3' H N O N O 3',5'-Phosphodiester 5' O P O CH2 O linkage O– 4' Adenine 2' NH2 3' H N N O N N 3',5'-Phosphodiester 5' O P O CH2 O linkage O– 4' 2' 3' H OH 3' end Specifying the primary structure for a nucleic acid is done by listing nucleotide base components (using their one-letter abbreviations) in sequential order starting with the base at the 59 end of the nucleotide strand. The primary structure for the four-nucleotide DNA segment shown in Figure 22.5 is 59 T–G–C–A 39 Three parallels between primary nucleic acid structure and primary protein structure (Section 20.10) are worth noting: 1. DNAs, RNAs, and proteins all have backbones that do not vary in structure (see Figure 22.6). 2. The sequence of attachments to the backbones (nitrogen bases in nucleic acids and amino acid R groups in proteins) distinguishes one Figure 22.6 A comparison of the general primary structures of 5' end Different bases 3' end A nucleic acids and proteins. nucleic Base1 Base2 Base3 acid Phosphate Sugar Phosphate Sugar Phosphate Sugar N-terminal end Different C-terminal end R Groups A protein R1 O R2 O R3 O R4 O NH CH C NH CH C NH CH C NH CH C  22.4 Primary Nucleic Acid Structure 805 C H E MISTRY AT A G L A NC E Nucleic Acid Structure NUCLEIC ACID STRUCTURE Base Base Base Phosphate Sugar Phosphate Sugar Phosphate Sugar Nucleotide Nucleotide Nucleotide DEOXYRIBONUCLEIC ACID (DNA) RIBONUCLEIC ACID (RNA) Nucleotide Components Nucleotide Components Sugar Phosphate Sugar Phosphate HOCH2 O HOCH2 O O– O– O P OH O P OH OH OH O– O– OH H OH OH -D-2'-Deoxyribose Hydrogen phosphate ion -D-Ribose Hydrogen phosphate ion Bases Bases NH2 O NH2 O CH3 H H N N N N N N N N N O N N N O H H H H Adenine (A) Thymine (T) Adenine (A) Uracil (U) O NH2 O NH2 H H N N N N N N N N NH2 N O N N NH2 N O H H H H Guanine (G) Cytosine (C) Guanine (G) Cytosine (C) POSSIBLE NUCLEOTIDES POSSIBLE NUCLEOTIDES dAMP dGMP dCMP dTMP AMP GMP CMP UMP DNA from another, one RNA from another, and one protein from another (Figure 22.6). 3. Both nucleic acid polymer chains and protein polymer chains have direction- ality; for nucleic acids, there is a 59 end and a 39 end, and for proteins, there is an N-terminal end and a C-terminal end. The Chemistry at a Glance feature above summarizes important concepts relative to the makeup of the nucleotide building blocks (monomers) present in polymeric DNA and RNA molecules. 806 Chapter 22 Nucleic Acids 22.5 The DNA Double Helix Like proteins, nucleic acids have secondary, or three-dimensional, structure as well as primary structure. The secondary structures of DNAs and RNAs differ, so they will be discussed separately. The amounts of the bases A, T, G, and C present in DNA molecules were the key to determination of the general three-dimensional structure of DNA mol- ecules. Base composition data for DNA molecules from many different organ- isms revealed a definite pattern of base occurrence. The amounts of A and T were always equal, and the amounts of C and G were always equal, as were the amounts of total purines and total pyrimidines. The relative amounts of these base pairs in DNA vary depending on the life form from which the DNA is obtained. (Each animal or plant has a unique base composition.) However, the relationships %A 5 %T and %C 5 %G always hold true. For example, human DNA contains 30% adenine, 30% thymine, 20% guanine, and 20% cytosine. In 1953, an explanation for the base composition patterns associated with DNA molecules was proposed by the American microbiologist James Watson and the English biophysicist Francis Crick. Their model, which has now been validated in numerous ways, involves a double-helix structure that accounts for the equality of bases present, as well as for other known DNA structural data. The DNA double helix involves two polynucleotide strands coiled around each other in a manner somewhat like a spiral staircase. The sugar–phosphate backbones of the two polynucleotide strands can be thought of as being the out- side banisters of the spiral staircase (Figure 22.7). The bases (side chains) of each Figure 22.7 Three views of the Hydrogen DNA double helix. bonds 5' 3' G C T A C G A T G C T A T A Sugar–phosphate backbone T A T A C G G C A T A T 5' 3' Base pair a A schematic drawing that b A space-filling model in c A top view of the emphasizes the hydrogen which one DNA strand is double helix. bonding between bases on blue and the other strand the two chains. is orange. The bases are shown in lighter shades of blue and orange.  22.5 The DNA Double Helix 807 backbone extend inward toward the bases of the other strand. The two strands are connected by hydrogen bonds (Section 7.13) between their bases. Additionally, the two strands of the double helix are antiparallel—that is, they run in opposite directions. One strand runs in the 59-to-39 direction, and the other is oriented in the 39-to-59 direction. Base Pairing A physical restriction, the size of the interior of the DNA double helix, limits the The a-helix secondary structure of base pairs that can hydrogen-bond to one another. Only pairs involving one small proteins (Section 20.11) involves one polypeptide chain; the double- base (a pyrimidine) and one large base (a purine) correctly “fit” within the helix helix secondary structure of DNA interior. There is not enough room for two large purine bases to fit opposite each involves two polynucleotide chains. other (they overlap), and two small pyrimidine bases are too far apart to hydrogen- In the a-helix of proteins, the R bond to one another effectively. Of the four possible purine–pyrimidine combina- groups are on the outside of the tions (A–T, A–C, G–T, and G–C), hydrogen-bonding possibilities are most favorable helix; in the double helix of DNA, the bases are on the inside of the for the A–T and G–C pairings, and these two combinations are the only two that double helix. normally occur in DNA. Figure 22.8 shows the specific hydrogen-bonding interac- tions for the four possible purine–pyrimidine base-pairing combinations. The antiparallel nature of the two The pairing of A with T and that of G with C are said to be complementary. A polynucleotide chains in the DNA and T are complementary bases, as are G and C. Complementary bases are pairs of double helix means that there is a bases in a nucleic acid structure that can hydrogen-bond to each other. The fact that 59 end and a 39 end at both ends of complementary base pairing occurs in DNA molecules explains, very simply, why the double helix. the amounts of the bases A and T present are always equal, as are the amounts of G and C. The two strands of DNA in a double helix are complementary. This The two strands of DNA in a double helix are not identical—they are comple- means that if you know the order mentary. Complementary DNA strands are strands of DNA in a double helix with of bases in one strand, you can pre- base pairing such that each base is located opposite its complementary base. Wherever dict the order of bases in the other G occurs in one strand, there is a C in the other strand; wherever T occurs in one strand. a Figure 22.8 Hydrogen-bonding possibilities are more favorable when A–T and G–C base pairing occurs than when A–C and G–T base pairing occurs. (a) Two and C G three hydrogen bonds can form, T A respectively, between A–T and G–C base pairs. These combinations are present in DNA molecules. (b) Only one hydrogen bond can Thymine–Adenine Base Pairing Cytosine–Guanine Base Pairing form between G–T and A–C base (two hydrogen bonds form) (three hydrogen bonds form) pairs. These combinations are not present in DNA molecules. b T G A mnemonic device for recalling C A base-pairing combinations in DNA involves listing the base abbrevia- tions in alphabetical order. Then the first and last bases pair, and so do Thymine–Guanine Base Pairing Cytosine–Adenine Base Pairing the middle two bases. (only one hydrogen bond forms) (only one hydrogen bond forms) DNA: A C G T Carbon Oxygen Lone pair Hydrogen bond Another way to remember these base-pairing combinations is to note Nitrogen Hydrogen Attachment to backbone that AT spells a word and that C and G look very much alike. 808 Chapter 22 Nucleic Acids strand, there is an A in the other strand. An important ramification of this com- plementary relationship is that knowing the base sequence of one strand of DNA enables prediction of the base sequence of the complementary strand. The base sequence of a single strand of a DNA molecule segment is always written in the direction from the 59 end to the 39 end of the segment. 59 A–A–G–C–T–A–G–C–T–T–A–C–T 39 If the end designations for a base sequence (59 and 39) are not specified for a sequence of bases, it is assumed that the sequence starts with the 59 end base. In the base sequence A–C–G–T–T–C It is assumed that A is the 59 end base. E XAM P L E 22.1 Predicting Base Sequence in a Complementary DNA Strand Predict the sequence of bases in the DNA strand that is complementary to the single DNA strand shown. 59 C–G–A–A–T–C–C–T–A 39 Solution Because only A forms a complementary base pair with T, and only G with C, the com- plementary strand is as follows: Given: 59 C–G–A–A–T–C–C–T–A 39 Complementary strand: 39 G–C–T–T–A–G–G–A–T 59 Note the reversal of the numbering of the ends of the complementary strand compared to the given strand. This is due to the antiparallel nature of the two strands in a DNA double helix. Practice Exercise 22.1 Predict the sequence of bases in the DNA strand complementary to the single DNA strand shown. 59 A–A–T–G–C–A–G–C–T 39 Answer: 39 T–T–A–C–G–T–C–G–A 59 When generating a complementary base sequence from a given 59 to 39 base sequence, as was done in Example 22.1, the complementary base sequence ob- tained runs in the 39 to 59 direction because of the antiparallel relationship that exists between paired base sequences. Such 39 to 59 base sequences are acceptable as long as the directionality of the sequence is specifically noted. When needed, a 39 to 59 base sequence can be converted to a 59 to 39 base sequence by simply reversing the order of the bases listed. The following two base sequence notations are entirely equivalent to each other. 39 A-T-C-G 59 and 59 G-C-T-A 39 Hydrogen Bonding Interactions Hydrogen bonding between base pairs is an important factor in stabilizing the Hydrogen bonding is responsible DNA double helix structure. Although hydrogen bonds are relatively weak forces, for the secondary structure (double helix) of DNA. Hydrogen bonding each DNA molecule has so many base pairs that, collectively, these hydrogen bonds is also responsible for secondary are a force of significant strength. In addition to hydrogen bonding, base-stacking structure in proteins (Section 20.11). interactions also contribute to DNA double-helix stabilization.  22.6 Replication of DNA Molecules 809 Base-Stacking Interactions The bases in a DNA double helix are positioned with the planes of their rings parallel (like a stack of coins). Stacking interactions involving a given base and the parallel bases directly above and below it also contribute to the stabilization of the DNA double helix. These stacking interactions are as important in their stabiliza- tion effects as is the hydrogen bonding associated with base pairing—perhaps even more important. Purine and pyrimidine bases are hydrophobic in nature, so their stacking interactions are those associated with hydrophobic molecules—mainly London forces (Section 7.13). The concept of hydrophobic interactions has been encountered twice previously. Hydrophobic interactions involving the nonpolar tails of membrane lipids contribute to the structural stability of cell membranes (Section 19.10), and hydrophobic interactions involving nonpolar R groups of amino acids contribute to protein tertiary structure stability (Section 20.12). Use of the Term “DNA Molecule” The term DNA molecule is actually a misnomer, even though general usage of the term is common in news reports, in textbooks, and even in the vocabulary of scien- tists. It is technically a misnomer for two reasons. 1. Cellular solutions have pH values such that the phosphate groups present in the DNA backbone structure are negatively charged. This means DNA is actually a multicharged ionic species rather than a neutral molecule. 2. The two strands of DNA in a double-helix structure are not held together by covalent bonds but, rather, by hydrogen bonds, which are noncovalent inter- actions. Thus, double-helix DNA is an entity that involves two intertwined ionic species rather than a single molecule. Despite these considerations, usage of the term DNA molecule is accepted by most scientists and is used through the remainder of this textbook. 22.6 Replication of DNA Molecules DNA molecules are the carriers of genetic information within a cell; that is, they are the molecules of heredity. Each time a cell divides, an exact copy of the DNA of the parent cell is needed for the new daughter cell. The process by which new DNA molecules are generated is DNA replication. DNA replication is the biochem- ical process by which DNA molecules produce exact duplicates of themselves. The key concept in understanding DNA replication is the base pairing associated with the DNA double helix. DNA Replication Overview In DNA replication, the two strands of the DNA double helix are regarded as a pair of templates, or patterns. During replication, the strands separate. Each can then act as a template for the synthesis of a new, complementary strand. The result is two daughter DNA molecules with base sequences identical to those of the par- ent double helix. Details of this replication are as follows. Under the influence of the enzyme DNA helicase, the DNA double helix unwinds, and the hydrogen bonds between complementary bases are broken. This unwinding process, as shown in Figure 22.9, is somewhat like opening a zipper. The point at which the DNA double helix is unwinding, which is constantly changing (moving), is called the replication fork (Figure 22.9). The bases of the separated strands are no longer connected by hydrogen bonds. They can pair with free individual nucleotides present in the cell’s nucleus. As shown in Figure 22.9, the base pairing always involves C pairing with G and A pairing with T. The pairing process occurs one nucleotide at a time. After a free nu- cleotide has formed hydrogen bonds with a base of the old strand (the template), 810 Chapter 22 Nucleic Acids Figure 22.9 In DNA replication, 3' Old the two strands of the DNA double C G helix unwind, with the separated G A strands serving as templates for the Replication T C G C A T T C T formation of new DNA strands. Fork A G G A A Free nucleotides pair with the 3' Old T complementary bases on the G G 5' New G A C separated strands of DNA. T C G C C C T T G 3' New This process ultimately results in A G T T C T C the complete replication of the G A A A G C A A G A DNA molecule. C T G T C C T T C T 5' Old A A G A 5' Old the enzyme DNA polymerase verifies that the base pairing is correct and then cata- lyzes the formation of a new phosphodiester linkage between the nucleotide and the growing strand (represented by the darker blue ribbons in Figure 22.9). The DNA polymerase then slides down the strand to the next unpaired base of the tem- plate, and the same process is repeated. The net result of DNA replication is the production of two daughter DNA molecules, both of which are identical to the one parent DNA molecule from which they were formed. Figure 22.10 gives a “close-up” view of the relationships between parent DNA and the daughter DNA produced from it. Note that each daughter DNA molecule contains one strand from the parent DNA and one strand that is newly formed. The Replication Process in Finer Detail Though simple in principle, the DNA replication process has many intricacies. 1. The enzyme DNA polymerase can operate on a forming DNA daughter strand only in the 59-to-39 direction. Because the two strands of parent DNA run in opposite directions (one is 59 to 39 and the other 39 to 59; Section 22.4), only one strand can grow continuously in the 59-to-39 direction. The other strand must be formed in short segments, called Okazaki fragments (after their discoverer, Reiji Okazaki), as the DNA unwinds (Figure 22.11). The breaks or gaps in this daughter strand are called nicks. To complete the formation of this strand, the Okazaki fragments are connected by action of the enzyme DNA ligase. The strand that grows continuously is called the leading strand, and the strand that is synthesized in small segments is called the lagging strand. Figure 22.10 In DNA replication, AOGOCOTOTOA PARENT STRAND two daughter DNA molecules are produced from one parent DNA TOCOGOAOAOT NEWLY FORMED STRAND molecule, with each daughter DNA molecule containing one parent AOGOCOTOTOA DNA strand and one newly formed DNA strand. TOCOGOAOAOT SEGMENT OF PARENT DNA AOGOCOTOTOA NEWLY FORMED STRAND TOCOGOAOAOT PARENT STRAND SEGMENT OF DAUGHTER DNAs (both of which are identical and both of which contain one parent strand and one newly formed strand)  22.6 Replication of DNA Molecules 811 Direction of movement Replication fork Figure 22.11 Because the of replication fork enzyme DNA polymerase can act Leading strand only in the 59-to-39 direction, one 3' (grows continously) strand (top) grows continuously 5' in the direction of the unwinding, 3' and the other strand grows in Okazaki fragments segments in the opposite direction. Lagging strand 5' (grows in The segments in this latter chain segments) are then connected by a different 5' 3' enzyme, DNA ligase. 5' Nick 2. The process of DNA unwinding does not have to begin at an end of the DNA molecule. It may occur at any location within the molecule. Indeed, studies show that unwinding usually occurs at several interior locations simultaneously and that DNA replication is bidirectional for these locations; that is, it pro- ceeds in both directions from the unwinding sites. As shown in Figure 22.12, the result of this multiple-site replication process is the formation of “bubbles” of newly synthesized DNA. The bubbles grow larger and eventually coalesce, giving rise to two complete daughter DNAs. Multiple-site replication enables large DNA molecules to be replicated rapidly. Based on mode of action, several types of anticancer drugs exist. One large group of such drugs are the antimetabolites, drugs which are DNA-replication in- hibitors. The focus on relevancy feature Chemical Connections 22-A on page 813 discusses several substances that find use as DNA-replication inhibitors. The Chemistry at a Glance feature on the next page summarizes the steps, as discussed in this section, that occur in the process of DNA replication. Chromosomes Once the DNA within a cell has been replicated, it interacts with specific pro- teins in the cell called histones to form structural units that provide the most stable arrangement for the long DNA molecules. These histone–DNA complexes are called chromosomes. A chromosome is an individual DNA molecule bound to a Chromosomes are nucleoproteins. group of proteins. Typically, a chromosome is about 15% by mass DNA and 85% They are a combination of nucleic by mass protein. acid (DNA) and various proteins. Origins of replication Figure 22.12 DNA replication usually occurs at multiple sites Parent DNA within a molecule, and the replication is bidirectional from these sites. Early stage in replication Later stage in replication Daughter DNAs 812 Chapter 22 Nucleic Acids C H E MISTRY AT A G L A NC E DNA Replication STEP 2 Free nucleotides pair G with their complementary base on the template strands by means of hydrogen bonds. A C STEP 3 DNA polymerase joins T the newly attached nucleotides to create one continuous strand STEP 1 in the 5'-to-3' direction. The enzyme DNA helicase causes 3' (old) the two strands of DNA to unwind, producing two template strands. A A G T G G T G C T G A A T G G G T C A G C A 5' (old) T G A C C C G T A C C T 5' (new) G G G T C A G T A C A T T 3' (new) A 3' (old) T G G G A C G T C T A A C A G C A A G T A G A C C C T G C A T T C C 5' (old) STEP 4 The other strand is formed in short segments (Okazaki fragments) in the 3'-to-5' direction. The segments are then joined together by DNA ligase. Cells from different kinds of organisms have different numbers of chromo- © Erica Stone/Peter Arnold, somes. A normal human has 46 chromosomes per cell, a mosquito 6, a frog 26, a dog 78, and a turkey 82. Chromosomes occur in matched (homologous) pairs. The 46 chromosomes of a Inc./Photolibrary human cell constitute 23 homologous pairs. One member of each homologous pair is derived from a chromosome inherited from the father, and the other is a copy of one of the chromosomes inherited from the mother. Homologous chromosomes have similar, but not identical, DNA base sequences; both code for the same traits Figure 22.13 Identical twins share but for different forms of the trait (for example, blue eyes versus brown eyes). Off- identical physical characteristics spring are like their parents, but they are different as well; part of their DNA came because they received identical from one parent and part from the other parent. Occasionally, identical twins are DNA from their parents. born (Figure 22.13). Such twins have received identical DNA from their parents.  22.6 Replication of DNA Molecules 813 C HE MICAL CONNECTIONS 22-A Antimetabolites: Anticancer Drugs That Inhibit DNA Synthesis Cancer is a disease characterized by rapid uncontrolled cell division. Rapid cell division necessitates the synthesis of large amounts of DNA, as DNA must be present in each new cell produced. Numerous anticancer drugs are now © Li Wa/Shutterstock.com available that block DNA synthesis and therefore decrease the rate at which new cancer cells are produced. Antimetabolites are a class of anticancer drugs that in- terfere with DNA replication because their structures are similar to molecules required for normal DNA replication. The structural similarity is close enough that enzymes can be “tricked” into using the drug rather than the real substrate needed. This “trickery” shuts down DNA synthesis, which A cancer patient undergoing chemotherapy causes cells to die. to inhibit DNA synthesis. Four examples of commonly used antimetabolites and 4. Methotrexate: The previous three antimetabolites were all the molecules they “mimic” are as follows: close structural analogs of bases found in DNA nucleotides. 1. 6-Mercaptopurine (6-MP): 6-MP structurally resembles Some antimetabolites are not base analogs. Methotrexate is one adenine, one of the four nitrogen-containing bases present in of these non-base analogs. It is a structural analog of folic acid all DNA molecules. Synthesis of adenine-containing nucleo- (folate), which is one of the B vitamins (Section 21.14). A deriv- tides is inhibited when 6-MP is present; nonfunctional DNA ative of folic acid is needed in one of the early steps of nucleo- results when 6-MP, rather than adenine, is incorporated into tide synthesis. Methotrexate inhibits the conversion of folic acid a nucleotide. to this needed derivative, which shuts down DNA synthesis. SH NH2 N N A A CH3 ENH2 N N A N N N N N A N N N N H D D HOOCH N NH2 H H A B 6-Mercaptopurine Adenine HOOC O (a modified adenine) Methotrexate 2. Thioguanine: As was the case with 6-MP, the close struc- N N tural resemblance between thioguanine and guanine leads to H ENH2 the incorporation of thioguanine, rather than guanine, into A N N nucleotides. Nonfunctional DNA is the result. N A H OH SH O HOOCH N A B A B N EH N EH HOOC O N N Folic acid N H NH N H NH N 2 N 2 D D Note that all four of these antimetabolites are synthetically H H produced molecules rather than naturally occurring ones. Many Thioguanine Guanine (a modified guanine) different synthetically modified purine and pyrimidine deriva- tives are now available for use in studies that involve DNA. 3. 5-Fluorouracil: Uracil is a base found in RNA rather than While some types of cancer respond very well to chemo- DNA. However, its structure is close enough to that of thy- therapy using antimetabolite drugs (leukemia is one such mine (which is methyluracil) that it can pass for thymine. cancer), other types of cancer do not respond well to such It thus inhibits the synthesis of active thymine-containing treatment. Two other general types of anticancer drugs nucleotides needed for DNA synthesis. are available for use in these treatment situations. They are (1) DNA-damaging agents and (2) cell-division inhibitors. Cis- O O platin is a well-known DNA damaging agent, and the natural B B products vincristine (obtained from the periwinkle plant) and FH EH CH3H EH N N taxol (obtained from yew tree bark) are cell-division inhibitors. NO NO Cells most affected by anticancer drugs are those undergo- N N ing rapid cell division (the cancer cells). However, normal cells A A H H are also affected, to a lesser extent, by these drugs. Eventually, 5-Fluorouracil Thymine the normal cells are affected to such a degree that use of the (a modified thymine) drugs must be discontinued, at least for a period of time. 814 Chapter 22 Nucleic Acids 22.7 Overview of Protein Synthesis In the previous section it was shown how the replication of DNA makes it possible for a new cell to contain the same genetic information as its parent cell. How the genetic information contained in a cell is expressed in cell operation will now be considered. This leads to the topic of protein synthesis. The synthesis of proteins (skin, hair, enzymes, hormones, and so on) is under the direction of DNA mol- ecules. It is this role of DNA that establishes the similarities between parent and offspring that are regarded as hereditary characteristics. The overall process of protein synthesis is divided into two phases. The first