Nucleic Acids - Learning Material PDF

VI. NUCLEIC ACIDS The Swiss physiologist Friedrich Miescher (1844-1895) discovered nucleic acids in 1869 while studying the nuclei of white blood cells. The fact that they were initially found in cell nuclei and are acidic accounts for the name nucleic acid. It is now known that nucleic acids are f...

VI. NUCLEIC ACIDS The Swiss physiologist Friedrich Miescher (1844-1895) discovered nucleic acids in 1869 while studying the nuclei of white blood cells. The fact that they were initially found in cell nuclei and are acidic accounts for the name nucleic acid. It is now known that nucleic acids are found throughout a cell, not just in the nucleus. It is probable that life itself began and evolved with nucleic acids, the most fundamental constituents of the living cell. Of all biomolecules, it is only the nucleic acids that have the remarkable property of replicating itself, thus nature chose these molecules to serve as the repository and transmitter of genetic information in every cell and organism. The genome or total DNA of a cell acts like a molecular file where the program for an organism’s activities (maintenance, development, growth, reproduction, and even death) are encoded. The nucleic acids (DNA in particular) are the “informational molecules”; into their primary structure is encoded a set of directions that ultimately governs the metabolic activities of the living cell. - Gene is a segment of DNA which specifies the chain of amino acids that comprises the protein molecule - The genetic message is transcribed by mRNA and translated by tRNA and rRNA into thousands of different proteins. The Central Dogma: Composition of Nucleic Acids: NUCLEIC ACID Nucleotides Nucleosides Phosphoric acid Bases: Sugars: a) pyrimidines: C, U, T a) β – D – ribose b) purines: G, A b) β – D – 2’ – deoxyribose A. Bases (nitrogen-containing heterocyclic bases) - are heterocyclic amines which are weak bases; have common and rare tautomers 1) Pyrimidines (monocyclic base with a six-membered ring) - include Cytosine, Thymine, and Uracil 2) Purines (bicyclic base with fused five- and six-membered rings); pyrimidine ring fused to imidazole ring; - include Adenine and Guanine 1 B. Sugars C. Nucleosides : Base + Sugar - the linkage is formed between C1’ of sugar and N1 of pyrimidine or N9 of purine base - the nucleosides of ribose with the bases are called: Cytidine, Thymidine, Uridine, Guanosine, and Adenosine - if deoxyribose is present, the prefix deoxy is used; eg, deoxyuridine (dU) - in numbering the atoms in nucleosides, a superscript prime is used to denote the atoms in the pentose D. Nucleotides : Nucleosides + H3PO4 - the basic subunits of the nucleic acids; - are phosphoesters of the nucleosides; - the linkage is formed between H3PO4 and C5’ of nucleoside - the phosphate group makes nucleotides strongly acidic - the terms “nucleotide” and “nucleoside phosphate” can be used 2 Names of the nucleotides 1) The 5’-nucleoside monophosphate of ……… is called ………. a) adenosine ………. adenylic acid or adenosine monophosphate (AMP) b) guanosine ………. guanylic acid or guanosine monophosphate (GMP) c) cytidine ………… cytidylic acid or cytidine monophosphate (CMP) d) uridine ………….. uridylic acid or uridine monophosphate (UMP) e) thymidine ………. thymidylic acid or thymidine monophosphate (TMP) 2) The 5’-nucleoside diphosphates are ADP, GDP, CDP, UDP, TDP 3) The 5’-nucleoside triphosphates are ATP, GTP, CTP, UTP, TTP E. Polynucleotides and the Nucleic Acids - the nucleotides of a polynucleotide chain are linked to one another in 3’, 5’-phosphodiester bonds ; phosphoric acid forms a phosphate ester to connect the 3’ hydroxyl group of one pentose to the 5’ carbon on another pentose 3 Shorthand Structure of Polynucleotides - bases are indicated by their initials, the ribose by a straight line extending from the base, and the phosphate by P. The C3’ and C5’ of the ribose or deoxyribose are indicated by the fact that the C5’ is at the end of the ribose line and the C3’ is toward the middle of the line. Thus if adenosine and guanosine are joined by a phosphate from the C3’ of adenosine to the C5’ of guanosine it can be represented as: Takadiasase (mold)  attacks “b” linkages in which “a” is linked to a purine nucleotide RNAse (bovine pancreas)  attacks “b” linkages in which “a” is linked to a pyrimidine nucleotide 4 I. Deoxyribonucleic acid (DNA) - a high mol.wt., double-stranded polynucleotide that occurs almost exclusively in the nucleus of the cell - the primary structure of DNA, referring to the sequence of the nucleotides in the chain, has individuality. By convention, the sequence is written and read in the 5’ to 3’ direction (polarity). Genetic information is encoded in the primary structure of the DNA - its primary function is the storage and transfer of genetic information which is used (indirectly) to control many functions of a living cell - the base content of DNA displays three sets of equivalent pairs: a) A + G = T + C (purine/pyrimidine ratio = 1) b) A = T c) G = C - the structure of the four bases permit hydrogen bonding between specific base pairs: Adenine always pairs with Thymine and Guanine with Cytosine ; Adenine forms 2 H bonds with thymine A=T ; Cytosine forms 3 H bonds with guanine G=C - the proof of this base-pairing came when Watson and Crick proved by x-ray diffraction that the DNA structure was a double helix whose chains were complementary and antiparallel complementary  means that A binds to T and C to G between the chains; the sequence of bases on one strand automatically determines the sequence of bases on the other strand antiparallel  means that each end of the helix contains the 5’ end of one strand and the 3’ end of the other, so that the chains travel in opposite directions; only when the 2 strands are antiparallel can the base pairs form the H bonds that hold them together Features of the Crick-Watson model for DNA - the DNA molecule is double stranded, where the two complementary and antiparallel chains are coiled around one another in a right-handed double helix - the sugar-phosphate backbones of the two strands spiral around the outside of the helix like the handrails on a spiral staircase; the nitrogenous bases extend into the center at right angles to the acids of the helix as if they are the steps of the spiral staircase - the diameter of the double helix is 2.0 nm which distance is dictated by the dimensions of the purine-pyrimidine base pairs This helical repetitive or regular folding structure (secondary structure) is stabilized by a number of factors: a) Chargaff’s rule of base pairing : A – T (2 H-bonds) and G – C (3 H-bonds); one strand is complementary to the other; b) Stacking interaction of the bases (the purines and pyrimidine rings) c) Hydrophobic interior (bases) and hydrophilic exterior (sugar-phosphate backbone) ; contact with bases through spiral grooves : major and minor grooves 5 Conformations of DNA: (p 468, McKee) DNA can assume different conformations because deoxyribose is flexible and the C1’ – N- glycosidic linkage rotates. (Recall that furanose rings have puckered conformation) a) B-DNA – the common form as described by Watson and Crick model b) A-DNA – when DNA becomes partially dehydrated it assumes the A-form; observed when DNA is extracted with solvents such as ethanol. c) Z-DNA – named for its “zigzag” conformation; DNA segments with alternating purine and pyrimidine bases (esp. CGCGCG) are most likely to adopt a Z configuration; regions of DNA rich in GC repeats are often regulatory, binding specific proteins that initiate or block transcription. Denaturation of DNA The loss of helical structure due to disruption of H–bonds is called denaturation or melting, where the double strands separate into single strands. This can be due to extremes of pH, heat, or chemicals that disrupt H-bonds. DNAs which are G-C rich denature at a higher temperature (Tm) than those which are A-T rich. Types of DNA sequences: 1. Exons – the coding sequences; interrupted by noncoding sequence 2. Introns – the noncoding sequences; from 10 to 10,000 bases long - removed in the interpretation to form proteins; when DNA of eukaryotic cell is transcribed, the coding due to introns are removed 6 3. Palindrome or inverted repeats (p 468, McKee) - a DNA sequence that contains the same information whether it is read forward or backward; e.g. “MADAM, I’M ADAM”; tendency to form hairpin loop and a snapback (cruciform); perfect palindrome forms with exact base pairs; quasi palindrome, when not all will form hairpin loop 4. Cruciform (or snapback) - as their name implies, are crosslike structures - likely to form when a DNA sequence contains a palindrome 5. Triple helix (also referred to as H-DNA; Hoogsteen base pairing) 6. Transposons - mobile/jumping genes 7. Retroposons - intronless genes; all coding genes; flanked by palindromes 7  Functions of the DNA As carrier of genetic information DNA serves two main functions: to make exact copies of itself in the process of replication or duplication; and to pass on the information coded in it to mRNA in the process of transcription so that mRNA in turn may translate the information in the 4-letter language of the nucleic acids. A. DNA Replication - the double-stranded DNA separates (opening of the helix is due to the enzyme helicase which breaks H-bonds; base pairs are separated, 3’ from 5’) and each half picks up new nucleotides using complementary base pairing. DNA polymerase forms sugar-phosphate bonds between the aligned nucleotides to complete the backbone, and H- bonds hold the base pairs together. When the process is completed two identical molecules of DNA have formed. - DNA replication is semiconservative and mostly bidirectional. - first step is the separation of the strands a) accomplished by helicase, which breaks the hydrogen bonds between base pairs b) positive supercoiling results when hydrogen bonds are broken, this is relieved by topoisomerase c) when supercoiling is relieved, single-strand binding protein binds to the separated strands to keep them apart d) primase catalyzes synthesis of a 10-12 base piece of RNA to “prime” the DNA replication - after the first step is completed, DNA polymerase III “reads” the parental strand or template, catalyzing the polymerization of a complementary daughter strand - Requirements: a) template b) a primer c) dNTPs (dATP, dTTP, dCTP, dGTP) d) Mg2+ - there are different mechanisms for replication of the two strands the leading strand is replicated continuously the opposite strand, the lagging strand, is replicated in segments, or discontinuously - the 3’ strand is called the leading strand because it is replicated in a continuous process; the 5’ strand is the lagging strand because it is replicated in a discontinuous mechanism. In the Leading Strand: The DNA Polymerase requires not only dNTP’s and Mg2+, but also a 5’-3’ orientation which can be offered only by the 3’strand, the leading strand. The enzyme cause the replication by base-pairing the 3’strand (as template) with free nucleotide units. Two conditions must be satisfied for replication to take place with high fidelity and accuracy: a) normal electronic characteristics b) normal base sequence 8  In the Lagging Strand The enzyme primase (using NTP’s: ATP, GTP, UTP, CTP and Mg2+) puts primers on the lagging strand by forming short RNA strands by base-pairing the 5’strand (template). Once primers have been formed, the DNA Polymerase will lengthen the primers using dNTP’s. The primers are then removed by enzyme nucleotidase and further lengthening is done by DNA Polymerase resulting to an OKAZAKI STRAND. The Okazaki strands are then linked together and sealed using the enzyme ligase leading to the formation of a NEW STRAND. 2) Transcription - biosynthesis of RNA by a DNA-dependent RNA Polymerase on a DNA template; an information transfer process where one of the two DNA strands acts as a template, which is copied into a complementary RNA molecule - transcription of DNA into RNA is restricted to discrete regions of DNA; DNA that has a code is transcribed to RNA, if specific code it is transcribed to mRNA - when a gene is transcribed, only one strand of the DNA serves as the template for RNA synthesis: the template strand, which is also called the minus (-) strand or sense strand; the nontemplate strand is called the coding strand, also called the plus (+) strand or antisense strand. - the bottom (blue) strand in the example below is the template strand, or the minus (-) strand, or the sense strand. The enzyme RNA polymerase sythesizes an mRNA in the 5' to 3' direction complementary to this template strand. The opposite DNA strand (red) is called the coding strand, the nontemplate strand, the plus (+) strand, or the antisense strand. The easiest way to find the corresponding mRNA sequence (shown in green) is to read the coding, or antisense strand directly in the 5' to 3' direction substituting U for T. 5' T G A C C T T C G A A C G G G A T G G A A A G G 3' 3' A C T G G A A G C T T G C C C T A C C T T T C C 5' 5' U G A C C U U C G A A C G G G A U G G A A A G G 3' - RNA Polymerase recognizes promoter sites (sequence of bases which signals where to start) and enhancer sites (base sequence which make recognition clearer) on DNA. These sites interact to define the region for transcription. Like DNA Polymerase, RNA Polymerase requires a 5’-3’ direction which can only be provided by the 3’strand of DNA; the RNA Polymerase, once it has spotted the portion to be transcribed, does the transcription in the 5’  3’ direction and uses NTP’s and Mg2+ 9 - Eukaryote mRNA is a primary transcript which still must be processed in post-transcriptional modification, a three step process: A 5' cap structure is added  this structure is required for efficient translation of the final mRNA A 3' poly(A) tail (100 to 200 units) is added by poly(A) polymerase  poly(A) tail protects the 3' end of the mRNA from enzymatic digestion  prolongs the life of the mRNA RNA splicing is the removal of portions of the primary transcript that are not protein coding  the introns are cut out and the exons (coding sequences) are spliced together - the genetic information is transcribed into mRNA by complementary base pairing and the exons in DNA is transcribed as the codon in mRNA - transcripts due to introns are removed by self-splicing through spliceosomes, which are composed of “small nuclear ribonucleoproteins (snRNPs, read “snurps”) ; the transcripts due to exons are joined by ligase 10 Replication and Transcription occur with very high accuracy and fidelity (normal base sequence and normal electronic character) except under conditions of spontaneous and induced mutation Once the mRNA is formed and released from DNA, it moves into the cytoplasm and combines with rRNA in ribosomes where protein synthesis occurs. II. Ribonucleic Acid (RNA) - a single-stranded polynucleotide with ribose as its pentose; occurs in all parts of a cell. - differs from DNA in the following: contains ribose ; has uracil instead of thymine ; single-stranded ; purine/pyrimidine ratio is not 1:1 - base pairing between U and A and G and C can still occur; this H bonding results in portions of the single-strand that become double-stranded - functions primarily in the synthesis of proteins, the molecules that carry out essential cellular functions Types of RNA - distinctions are made primarily on the basis of biochemical function ; differ in molar mass and 2o structure 1) messenger RNA (mRNA) - transcripts of certain segments of DNA; information-carrying intermediates in protein synthesis - shown to be complementary to a given segment of the DNA of the organism from which it is isolated - synthesized using DNA as a template in a process known as transcription. (transcription because the mRNA is a complementary copy of the information contained in the DNA - molecular dimensions vary according to the amount of genetic information that the molecule is meant to encode - directly governs protein synthesis ; has high affinity for the small subunit ribosomes, the active sites of protein synthesis 2) transfer RNA (tRNA) - relatively low molar mass nucleic acid ; ~27 – 90 nucleotides - functions by attaching itself, with the aid of a specific enzyme to a particular amino acid and by carrying that amino acid to the site of protein synthesis at the precise moment specified by the genetic code - each of the 20 aa’s found in proteins has at least one corresponding tRNA ; most aa’s have multiple tRNA molecules; - tRNAs act as “adaptor” molecules to translate mRNA language to amino acids via the genetic code - complementary base pairings also explains the different conformations of single-stranded RNA - loops are formed in the clover leaf conformation of tRNA because at some points H-bonded base-pairs are possible 3) ribosomal RNA (rRNA) - comprises 85-90% of the total cellular component of RNA; constitutes about 60% of the ribosomes (40% protein) - structurally composed of two spherical particles of unequal size: the smaller has affinity for mRNA ; the larger has an attraction for tRNA ; provide a space, “workbench” for protein synthesis - the tRNA and the rRNA are not translated, only the mRNA 11 The Genetic Code “How can a molecule (DNA) with just four different monomeric units specify the sequence of the 20 different amino acids that occur in proteins?” - if each nucleotide coded for one different amino acid, then obviously the nucleic acids could code for only 4 of the 20 amino acids. Suppose we consider the nucleotides in groups of two : there are 42 or 16 different combinations of pairs of the four distinct nucleotides. Such a code is more extensive but is still inadequate. If, however, the nucleotides are considered in groups of three : there are 43 or 64 different combinations. Here we have a code that is extensive enough to govern the 1o structure of the protein molecule because it contains more than enough coding units to designate all 20 amino acids - because the code involves 3 bases per coding unit it is referred to as a triplet code. The coding unit is called a codon. The genetic code is a series of base triplets in mRNA called codons that code for a particular amino acid. Cracking the Code : - early experiments were faced with the task of determining which codon (or codons) stood for each of the 20 amino acids; the cracking of the genetic code was a joint accomplishment of several well-known scientists, notably Khorana, Nirenberg, Leder, & Ochoa. A fairly extensive genetic dictionary was compiled with the ff. features: nonoverlapping: no bases are shared between consecutive codons commaless: no intervening bases between codons ; codons are written in a 5' 3' sequence degenerate: more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets universal: the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria 12 PROTEIN SYNTHESIS Overview: - the expression of the information contained in the DNA is fundamental to the growth, development, and maintenance of all organisms - if the sequence of bases along the DNA strand determines the sequence of amino acids along the protein chain, then the information contained in the DNA must be conveyed from the nucleus to the ribosomes, the site of protein synthesis. This is accomplished by phenomenon known as transcription (biosynthesis of mRNA) - once formed, the mRNA diffuses to the ribosomes carrying with it the genetic instructions. Each group of three bases along the mRNA strand now specifies a particular amino acid, and the sequence of these triplet groups dictates the sequence of the amino acids in the protein - now the cell faces the problem of lining up the amino acids according to the sequence called for by the mRNA, and of joining them together by means of peptide linkages. Because this process involves the transfer of the information encoded in the mRNA to the ultimate structure of the protein molecule, it is often referred to as translation (biosynthesis of protein) - unlike transcription, the process of translation involves converting the information from one language to another. In this case the genetic information in the linear sequence of nucleotides is being translated into proteins, a linear sequence of amino acids. Steps: (in eukaryotes) 1) Amino acid activation - before the amino acids may be incorporated into a polypeptide chain, they must first be activated. Activation occurs prior to the reaction of the amino acid with its particular tRNA carrier molecule. The amino acid combines with a molecule of ATP, yielding a compound known as aminoacyl adenylate. The reaction is enzyme-catalyzed - the aminoacyl adenylate remains on the surface of the enzyme and then undergoes reaction with the proper tRNA molecule to form the corresponding aminoacyl-tRNA complex (charged tRNA). Both the enzymes (aminoacyl synthetases) and the tRNA’s are highly specific for a particular amino acid. 13 2. Initiation - initiation factors (proteins), mRNA, initiator tRNA, and small and large ribosomes come together - ribosome has two sites to bind tRNA P-site binds to the growing peptide A-site binds the aminoacyl tRNA - binding of the initiator tRNA to the start signal of mRNA (AUG) at the P site of the ribosome - proteins called initiation factors, IF, are required to mediate the formation of a translation complex composed of an mRNA molecule, small and large subunit of ribosomes, and the initiator tRNA. The initiator tRNA recognizes the initiation codon, AUG. - formation of an initiation complex sets protein synthesis in motion. The mRNA and IF bind to small ribosomal subunit (30S). Next, a methionyl-charged tRNA (met-tRNA) binds, and finally the IF are released and the large ribosomal subunit (50S) binds to form the complete, functional 70S ribosome. 3. Elongation - elongation occurs in three steps that are repeated until protein synthesis is complete: a) first, the binding of the aminoacyl-tRNA to the empty A-site (amino acyl-tRNA binding site) b) next, peptide bond formation occurs catalyzed by an enzyme peptidyl transferase that is part of the ribosome. Now the peptide chain is shifted to the tRNA that occupies the A site. c) finally, the uncharged tRNA molecule left on the P site is discharged, and the ribosome changes position so that the next codon on the mRNA occupies the A-site. This movement is called translocation, which shifts the new peptidyl-tRNA from the A-site to the P-site. 14 4. Termination - the last stage of translation; UAA, UAG, & UGA are the termination codons for which there are no corresponding tRNA carrier molecules; upon finding a “stop” codon , a release factor, RF, binds the stop codon on mRNA on the empty A-site. This leads to the hydrolysis of the ester bond linking the peptide to the peptidyl-tRNA molecule in the P-site. The ribosome then dissociates into its two subunits, releasing the mRNA and the newly synthesized protein. - the protein released may not be in its final form; post-translational modifications may occur before a protein is fully functional: cleavage of f-met (initiation codon); association with other proteins; bonding to carbohydrate or lipid groups; S – S bonds between cys units 15 Regulation of Protein Synthesis When mRNA acts as a template for the synthesis of proteins, only a small amount of the total information in DNA is used at one time to produce a certain type of protein. What determines whether mRNA is formed from a certain segment of the DNA? What turns “ON” the DNA and what turns it “OFF”? Since protein is not synthesized continuously but only as needed, DNA must normally be in a “repressed state”. A repressor, which is a polypeptide, binds to small segment of the DNA. This segment is called the operator site. As long as the repressor is bonded to the DNA, no mRNA is produced and production of protein is inhibited. When a particular protein is needed, an inducer is formed. The inducer combines with the repressor, changing its shape so that it can no longer bind to the DNA. Once the repressor is removed from the DNA, syntheis of mRNA and hence protein can begin. When sufficient protein has been synthesized, the enducer is removed and the repressor once again binds to the DNA, stopping protein synthesis. Many Antibiotics Inhibit Protein Synthesis Several antibiotics stop bacterial infections by interfering with the synthesis of proteins needed by the bacteria. Some antibiotics act only on bacterial cells by binding to the ribosomes in bacteria, but do not act on human cells. Antibiotic Effect on ribosomes to inhibit protein synthesis Chloramphenicol Inhibits peptide bond formation and prevents the binding of tRNA’s Erythromycin Inhibits peptide chain growth by preventing the translocation of the ribosome along the mRNA Puromycin Causes release of an incomplete protein by ending the growth of the polypeptide early Streptomycin Prevents the proper attachment of tRNA’s; mRNA misreading by binding 30S Tetracycline Prevents the binding of tRNA’s by binding to 30S subunit ================================================================================= Replication, transcription, and translation occur with very high accuracy and fidelity (normal base sequence and normal electronic character) except under conditions of spontaneous and induced mutation Mutations (pp 462-464 McKee) - mutations are mistakes introduced into the DNA sequence of an organism; are microlesions and macrolesions of DNA; microlesions (small deletions) include transition, transversion, and frameshift mutation - macrolesions (large deletions) include repetitive sequence misalignment, palindromic misalignment, or both - mutations can be silent, that is, cause no change in the protein - many mutagens are also carcinogens and cause cancer, chemicals causing a change in the DNA sequence 16 Mechanisms of Spontaneous Mutations 1. Point Mutations - substitution of a single nucleotide for another - caused by tautomeric base-mispairs due to the ease by which rare tautomers are formed - C is the most mutable base due to the very small energy difference between its two tautomers - in nature, there are more A-T pairs than G-C pairs to protect us from the effect of spontaneous mutation A. Transition (C-A* ; G-T* ; C *- A mispair) - a purine base is changed to another purine; a pyrimidine base to another pyrimidine B. Transversion (A-A* mispair) - a purine base is changed to pyrimidine; a pyrimidine base changed to purine 2. Frameshift mutation - leads to a change in the reading frame A. Insertion - one or more nucleotides are added B. Deletion - one or more nucleotides are lost 1. Simple misalignment 2. Palindromic misalignment In an insertion or deletion mutation, a base is added to or deleted from the DNA sequence. Then a frameshift occurs which leads to a misreading of all the codons following the base change. 17 Cancer In an adult, most cells in the body do not continue to reproduce. When cells in the body begin to grow and multiply without control, they invade neighboring cells and appear as a tumor or growth (neoplasm). When tumors interfere with normal function of the body, they are cancerous. If they are limited, they are benign. Cancer can be caused by chemical and environmental substances, by radiation, or by oncogenic viruses. A carcinogen is any substance that increases the probability of inducing a tumor. It causes cancer by reacting with molecules in a cell (DNA), and altering the growth of that cell. Known carcinogens include dyes, cigarette smoke, and asbestos. Radiant energy from sunlight or from medical radiation is another type. Skin cancer results from mutations of the DNA of the skin cells exposed to these agents. The cells lose their ability to control proteins synthesis, and uncontrolled cell division leads to cancer. Oncogenic viruses cause cancer when the cells are infected. Induced Mutations - although a number of structural features of NA’s promote stabilization of base sequences, reactivity with some physical and chemical agents can alter the electronic characteristics of the bases and other structural units. Consequently, nucleic acid functions would be affected A. Physical agents of mutation include heat, UV, ionizing radiation (X-rays, -rays) - UV radiation falling on DNA is absorbed primarily by the pyrimidine bases. UV light causes covalent linkage of adjacent pyrimidine bases forming cyclobutane pyrimidine dimer when T is irradiated with UV, excitation of pi electrons to antibonding MO’s will result in the formation of T diradicals. Coupling of T diradicals may result in the formation of thymine cyclobutane dimers 18  Failure to repair this defect can lead to xeroderma pigmentosum; people who suffer from this genetic skin disorder are very sensitive to UV light and develop multiple skin cancers Pyrimidine dimer formation can be used to kill bacteria with UV exposure No purine dimers since purines are more thermodynamically stable than pyrimidines - heat mutagenesis characterized by transmigration of N – C glycosidic bonds producing neoguanosine crosslinks O N NH O N N NH2 -O P O O O- H H H H OH OH - ionizing radiation more often when a plant or animal is irradiated most of the energy is deposited in the aqueous phase. Less often will a primary ionization occur in an organic molecule a portion of damage to the living system results from reactive particles that are formed in the water phase and diffuse to an organic molecule in the cell causing secondary reactions (free radicals are implicated in radiation damage) 19 B. Chemical agents of mutation include alkylating agents, intercalating agents (PAH), heavy metal ions, etc. - one notorious source of numerous mutagens is cigarette smoke. It contains PAH, nitrosamines, hydrazines, pyrolysates, alkoxy free radicals, superoxide anion radicals, Cd2+, etc. - other notorious sources are: cured foods; burnt portion of broiled fish and meat; moldy peanuts and cereals; pesticides; polluted air (epoxides, SO2, ozone, Pb2+, ethylene dibromide, etc.) laboratory chemicals (benzene has been linked to leukemia, CHCl3, CCl4, etc) - intercalating agents - intercalating agents, like PAH (with polycyclic planar structures), interpose between the - strands within the grove of DNA, thereby inhibiting its replication/transcription, or cause deletions - bases in DNA are nucleophiles and as such are strongly attracted to electrophilic compounds; many carcinogens are electrophilic - alkylating agents – have electrophilic sites or may be metabolized to electrophiles which can interact with alkylating sites at DNA; include PAH, nitrosamines, aflatoxins, aromatic amines, epoxides, nitrogen mustard, nitrosoureas, etc. major alkylation sites at DNA bases:  O6 of G  N7 of G  O2 & O4 of T N7- alkylation leads to apurinic sites O6- alkylation leads to base mispairs 20 Figure. Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine. (b) Nitrosoamines, organic compounds that react to form nitrous acid, also lead to the oxidative deamination of A and C. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine. The result is a C-G to T-A transition. (d) Alkylation of G residues to give O6-methylguanine, which base-pairs with T. (e) Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards. Note that nitrosoamines are mutagenic in two ways: they can react to yield HNO2 or they can act as alkylating agents. The nitrosoguanidine, N-methyl-N'-nitro-N-nitrosoguanidine, is a very potent mutagen used in laboratories to induce mutations in experimental organisms such as Drosophila melanogaster. 21  What happens to the cell when a mutagen reacts with the DNA? A mutation is an alteration in the DNA base sequence that changes the structure and function of a protein in the cell. Some mutations are known to result from x-rays, overexposure to sun (UV light), chemicals called mutagens, and possibly some viruses. If a change in DNA occurs in a cell other than a reproductive cell, that altered DNA will be limited to that cell and its daughter cells and will not cause much harm. However, if the mutation occurs in the DNA of eggs or sperm, then all the DNA produced in a new individual will contain the same genetic error. If the mutation greatly affects the catalysis of metabolic reactions or the formation of important structural proteins, the new cells may not survive. Mutagen DNA lesions repaired cell death lesions that escape repairs in in in Somatic Cells Cells during Organogenesis Germ Cells CANCER BIRTH DEFECTS STERILITY GENETIC DISORDER (that can be transmitted from one generation to the next) Mutation effects may not be obvious for a long time. It is likely that the major effects of increased mutation rates would be spread insidiously over many generations and would include ill-defined abnormalities such as premature aging, increased susceptibility to various diseases including leukemia. Some chemical and environmental carcinogens Carcinogen Tumor occurrence Asbestos Lung, respiratory tract Arsenic Skin, lung Cadmium Prostate, kidneys Chromium Lung Nickel Lung, sinuses Aflatoxin Liver Nitrites Stomach Aniline dyes Bladder Vinyl chloride Liver Benzene Leukemia 22 Human cancers caused by oncogenic viruses Virus Disease RNA viruses Human T-cell leukemia-lymphoma virus-1(HTLV-1) Leukemia Human immunodeficiency virus (HIV) Acquired immune deficiency (AIDS) DNA viruses Epstein-Barr virus (EBV) Burkitt’s lymphoma (cancer of wbc) Nasopharyngeal carcinoma Hodgkin’s disease Hepatitis B virus (HBV) Liver cancer Herpes simplex virus (type 2) Cervical and uterine cancer Papilloma virus Cervical and colon cancer, genital warts DNA Repair (pp 499-500 McKee) DNA is the only macromolecule that is repaired rather than degraded. The repair processes are very efficient with fewer than 1 out of 1,000 accidental changes resulting in mutations. The rest are corrected through various repair mechanisms before, during, or after replication a) photoreactivation repair using an enzyme photolyase, binds the T-T cyclobutane dimer & in the presence of visible light changes the cyclobutane ring back into individual pyrimidine bases b) in excision repair, mutations are excised by a series of enzymes that remove incorrect bases and replace them with the correct ones. i. Base excision repair – a very important repair pathway involves a battery of enzymes called DNA glycosylases each of which recognizes a single type of altered base in DNA and catalyzes its hydrolytic removal from the deoxyribose sugar. At least 20 different such enzymes are thought to exist (e.g., removing deaminated cytosine, deaminated adenine, alkylated bases, etc.) ii. Nucleotide excision repair 23 24 Recombinant DNA and Genetic Engineering Techniques now exist whereby a “foreign” gene can be added to an organism, and the organism will produce the protein associated with the added gene. This technology is called genetic engineering or biotechnology. Genetic engineering procedure involves a type of bacteria called recombinant DNA, DNA that has been synthesized by splicing a segment of DNA (usually a gene) from one organism into the DNA of another organism. The case of human insulin is an example of the benefits that come from genetic engineering. For many years, because of the very limited availability of human insulin, the insulin used by diabetics was obtained from the pancreas of slaughterhouse animals. Such insulin is structurally very similar to human insulin. Today, diabetics use “real” human insulin produced by genetically altered bacteria. Such “genetically engineered” bacteria are grown in large numbers, and the insulin they produce is harvested in a manner similar to the way some antibiotics are obtained from cultured microorganisms. Human growth hormone is another substance that is now produced by genetically altered bacteria. Theory and Procedures The bacterium E. coli, which is found in the intestinal tract of humans and animals, is the organism most often used in recombinant DNA experiments. In addition to their chromosomal DNA, E. coli (and other bacteria) contains DNA in the form of small, circular, double-stranded molecules called plasmids. These plasmids, which carry only a few genes, replicate independently of the chromosome. Also, they are transferred relatively easily from one cell to another. 1. E. coli cells of a specific strain are placed in a solution that dissolves cell membranes, thus releasing the contents of the cells. 2. The released cell components are separated into fractions, one fraction being the plasmids, which is isolated and used in further steps. 3. A special enzyme, called restriction enzyme, is used to cleave the double stranded DNA of a circular plasmid. Restriction enzymes are bacterial enzymes that “cut” the sugar-phosphate backbone of DNA at specific nucleotide 25  sequences. An example of this type of enzyme is EcoRI, which cuts the DNA in a staggered fashion, cutting between the G and the first A of both strands, resulting in two DNA fragments. The staggered termini are called sticky ends as they can reassociate with one another by hydrogen bonding. 4. The same restriction enzyme is then used to remove a desired gene from a chromosome of another organism. If the same restriction enzyme used to cut a plasmid is also used to cut a gene from another DNA molecule, the sticky ends of the gene will be complementary to those of the plasmid. This enables the plasmid and gene to combine readily, forming a new, modified plasmid molecule; this is the recombinant DNA. 5. The gene (from step 4) and the opened plasmid (from step 3) are mixed in the presence of the enzyme DNA ligase, which splices the two together. This splicing, which attaches one end of the gene to one end of the opened plasmid and attaches the other end of the gene to the other end of the plasmid, results in an altered circular plasmid (the recombinant DNA). In addition to the newly spliced gene, the recombinant DNA plasmid contains all of the genes and characteristics of the original plasmid. 6. The altered plasmids (recombinant DNA) are placed in a live E. coli culture, where they are taken up by the E. coli bacteria. The E. coli culture into which the plasmids are placed need not be identical to that from which the plasmids were originally obtained The process in step 6 that involves inserting the recombinant DNA (modified plasmids) back into E. coli cells is called transformation. Transformation is the process of incorporating foreign DNA into a host cell. The transformed cells then reproduce, resulting in large numbers of identical cells called clones. Clones are cells that have descended from a single cell and have identical DNA. Within a few hours, a single genetically altered bacterial cell can give rise to thousands of clones. Each clone has the capacity to synthesize the protein directed by the foreign gene that it carries. 26 So, what is cloning? Strictly speaking, it's making many copies of a gene—in the example above, E. coli is doing the cloning. However, the term cloning is more generally used to refer to the entire process of isolating and manipulating a gene. Dolly the cloned sheep contained the identical genetic material of another sheep. Thus, researchers refer to Dolly as a clone. Scientists in Scotland were the first to clone an animal, this sheep named Dolly. She later gave birth to Bonnie, the lamb next to her. Photograph showing a transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right). Animals that have acquired new genetic information as a consequence of the introduction of foreign genes are termed transgenic. The methodology involves the injection of plasmids carrying the gene of interest into the nucleus of an oocyte or fertilized egg, followed by implantation of the egg into a receptive female. 27 28