Nucleic Acids - BCH 201 PDF

Nucleic acids Nucleic acids are macromolecules like proteins, carbohydrates and lipids. It is essential for all known forms of life. They are abundant in all living things, where they encode, transmit and express genetic information. Nucleic acids are divided into:- DNA (deoxyribonucleic acid) and...

Nucleic acids Nucleic acids are macromolecules like proteins, carbohydrates and lipids. It is essential for all known forms of life. They are abundant in all living things, where they encode, transmit and express genetic information. Nucleic acids are divided into:- DNA (deoxyribonucleic acid) and - RNA (ribonucleic acid). Nucleic acids consist of polymer nucleotide monomers. Each nucleotide has three components: a nitrogenous base, 5-carbon sugar, and a phosphate group. Nucleic acids carry the genetic and hereditary information of the cell and all the necessary information needed for the cell to perform all the life processes. The ability to store and transmit genetic information from one generation to the three next is a fundamental condition for life. Structure of Nucleotides Nucleotides are the building blocks of Nucleic Acids Nucleotides have three characteristic components: a nitrogenous base, a pentose, and a phosphate. The molecule without the phosphate group is called a nucleoside. The nitrogenous bases There are two categories of nitrogenous bases in the nucleic acids:- Purines (two-ring structure and contain Adenine (A) and Guanine (G)- Pyrimidines (one ring and contain Thymine (T), Cytosine (C) and Uracil (U)- DNA contains Adenine, Guanine, Thymine and Cytosine- RNA contains Adenine, Guanine, Uracil and Cytosine  The pentose sugar Nucleic acids have two kinds of pentoses. The RNA contains ribose. The DNA contains deoxyribose (ribose missing oxygen at position C2).  Nomenclature nucleoside = sugar +base nucleotide = sugar + base + phosphate Table: Names of DNA Base Derivatives -------------------------------------- -------------------- -------------------------------------- Base Nucleoside 5\'-Nucleotide Adenine 2\'-Deoxyadenosine 2\'-Deoxyadenosine-5\'-monophosphate Cytosine 2\'-Deoxycytidine 2\'-Deoxycytidine-5\'-monophosphate Guanine 2\'-Deoxyguanosine 2\'-Deoxyguanosine-5\'-monophosphate Thymine 2\'-Deoxythymidine 2\'-Deoxythymidine-5\'-monophosphate Roles of functional nucleotides They are the constituents of DNA and RNA. The structure of every protein, and ultimately of every biomolecule and cellular component, is a product of informational codes programmed into the nucleotide sequence of a cell\'s nucleic acids. They are the energy currency in metabolic transactions (mostly ATP). They are essential chemical links in the response of cells to hormones and other extracellular stimuli, such as cyclic adenosine monophosphate (cAMP). They are structural components of an array of enzyme cofactors and metabolic intermediates (ex., NAD+, FAD, FMN) Nucleotides derivatives NAD+/ NADH NADP+/ NADPH FAD/ FADH2 FMN/ FMNH2 c AMP, c GMP Nucleotides derivatives; NAD+ & NADP+ Nicotinamide adenine dinucleotide (NAD+) has many roles in the cell: It is a coenzyme for many oxidoreductases. So, it carries electrons from one reactant to another. It acts as a precursor of the second messenger molecule cyclic ADP-ribose, It acts as a substrate for bacterial ligase. It consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base, and the other nicotinamide (vitamin B3). NADH is the reduced form of NAD^+^. NADP+ has many functions in cells (search for its functions) Nucleotide derivatives; FAD & FMN Flavin adenine dinucleotide (FAD) is a redox cofactor for many oxidoreductases. FAD can be converted between three redox states by accepting or donating electrons. It is composed of adenine and flavin (Vitamin B2) Flavin mononucleotide (FMN) is another cofactor as FAD FADH~2~ Nucleotides derivatives; cAMP, cGMP Cyclic adenosine monophosphate (cAMP) is an important second messenger in many biological processes. It is a derivative of adenosine triphosphate (ATP) The phosphate group attached to C5 form cyclic form with the --OH of C3, so the name cyclic AMP Function: It is used for intracellular signal transduction in many different organisms, such as transferring the effects of hormones that cannot pass through the plasma membrane into the inside of the cell (like glucagon and adrenaline). It is also involved in the activation of protein kinases. It binds to and regulates the function of some ion channels. DNA Primary structure: Description of Phosphodiester Bond DNA primary structure Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids The successive nucleotides of DNA and RNA are covalently linked through phosphate-group \"bridges,\" in which the 5-phosphate hydroxyl group of one nucleotide unit is joined to the 3 group of the next nucleotide, creating a phosphodiester linkage. The backbone of both DNA and RNA are hydrophilic due to the--OH, NH, phosphate and C=O groups negatively charged in neutral pH due to the phosphate groups. DNA primary structure: Phosphodiester linkage The DNA or RNA strands have two ends: 5\'- phosphate end and 3\' -OH end  NA primary structure: The covalent backbones of nucleic acids consist of alternating phosphate and pentose residues, and the nitrogenous bases may be regarded as side groups joined to the backbone at regular intervals. The secondary structure of DNA (double helix) DNA is the macromolecular structure that carries the genetic instructions for all biological processes. Most DNA molecules consist of two strands of deoxyribonucleotides forming a double helix. These two strands wound around the same axis in a right-handed manner. The bases forming the DNA are A, G, T and C. All nucleotides in each strand are bound by a phosphodiester bond (type of covalent bond). The two strands bind together by hydrogen bonds. Adenine (A) in one strand binds Thymine (T) in the other by two hydrogen bonds. Guanine (G) in one strand binds Cytosine (C) in the other by three hydrogen bonds. The two strands run in an antiparallel manner. DNA secondary structure: (cont.) double helix properties, base pairing, stabilising forces The DNA double helix, or duplex, is held together by two forces, -hydrogen bonding between complementary base pairs and -base-stacking interactions. The complementarity between the two strands of DNA is attributed to the hydrogen bonding between base pairs. The base-stacking interactions, which are largely nonspecific concerning the identity of the stacked bases, significantly contribute to the double helix\'s stability.  In DNA The number of purines (A+G) = The number of pyrimidines (T+C) Chargaff\'s rule- The number of Guanine (G) - The number of Adenine (A) = The number of Cytosine (C) = The number of Thymine (T) So, we can calculate the ratio of three bases depending on the known ratio of any one base Tertiary structure of DNA The detailed structure of the double helix, the shapes of DNA, the major and minor groove triplexes and other forms Tertiary structure refers to the locations of the atoms in three-dimensional space, considering geometrical and steric constraints. The two antiparallel polynucleotide chains of double-helical DNA are complementary to each other. Wherever adenine occurs in one chain, thymine is found in the other; Wherever guanine occurs in one chain, cytosine is found in the other. The hydrophilic backbones of alternating deoxyribose and phosphate groups are outside the double helix, facing the surrounding water. The furanose ring of each deoxyribose is in the C-2 endo conformation. Both strands\' purine and pyrimidine bases are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis. The two strands create a major groove and a minor groove.  **WORKED EXAMPLE** Base Pairing in DNA In samples of DNA isolated from two unidentified species of bacteria, X and Y, adenine makes up 32% and 17%, respectively, of the total bases. What relative proportions of adenine, guanine, thymine, and cytosine would you expect to find in the two DNA samples? What assumptions have you made? One species was isolated from a hot spring (64 8C). Which species is most likely the thermophilic bacterium, and why? Solution: For any double-helical DNA, A 5 T and G 5 C. The DNA from species X has 32% A and, therefore, must contain 32% T. This accounts for 64% of the bases and leaves 36% as GqC pairs: 18% G and 18% C. The sample from species Y, with 17% A, must contain 17% T, accounting for 34% of the base pairs. The remaining 66% of the bases are thus equally distributed as 33% G and 33% C. This calculation is based on the assumption that both DNA molecules are double-stranded. The higher the G 1 C content of a DNA molecule, the higher the melting temperature. Species Y, having the DNA with the higher G 1 C content (66%), most likely is the thermophilic bacterium; its DNA has a higher melting temperature and thus is more stable at the temperature of the hot spring. DNA Can Occur in Different Three-Dimensional Forms DNA is a remarkably flexible molecule. Considerable rotation is possible around several types of bonds in the sugar-phosphate backbone, and thermal fluctuation can produce bending, stretching, and unpairing (melting) of the strands. Many significant deviations from the Watson-Crick DNA structure are found in cellular DNA, some or all of which may be important in DNA metabolism. These structural variations generally do not affect the key properties of DNA defined by Watson and Crick: strand complementarity, antiparallel strands, and the requirement for APT and GqC base pairs. Structural variation in DNA reflects three things: The different possible conformations of the deoxyribose rotation about the contiguous bonds make up the phosphodeoxyribose backbone and free rotation about the C-19--*N*-glycosyl bond. Because of steric constraints, purines in purine nucleotides are restricted to two stable conformations concerning deoxyribose, called syn and anti. Pyrimidines are generally restricted to the anti-conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 the pyrimidine. The Watson-Crick structure is also referred to as **B-form DNA**, or B-DNA. The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is, therefore, the standard point of reference in any study of the properties of DNA. Two structural variants that have been well characterised in crystal structures are the **A** and **Z** **forms**. The A form is favoured in many solutions that are relatively devoid of water. The DNA is still arranged in a righthanded double helix, but the helix is broader, and the number of base pairs per helical turn is 11 rather than 10.5 as in B-DNA. The plane of the base pairs in A-DNA is tilted about 20º relative to B-DNA base pairs. Thus, A-DNA\'s base pairs are not perpendicular to the helix axis. These structural changes deepen the major groove while making the minor groove shallower. The reagents used to promote the crystallisation of DNA tend to dehydrate it, and thus, most short DNA molecules tend to crystallise in the A form. Z-form DNA is a more radical departure from the B structure; the most apparent distinction is the left-handed helical rotation. There are 12 base pairs per helical turn, and the structure appears more slender and elongated. The DNA backbone takes on a zigzag appearance. Specific nucleotide sequences fold into left-handed Z helices much more readily than others. Prominent examples are sequences where pyrimidines alternate with purines, especially alternating C and G or 5-methyl-C and G residues. To form the left-handed helix in Z-DNA, the purine residues flip to the syn conformation, alternating with pyrimidines in the anti-conformation. The major groove is barely apparent in Z-DNA, and the minor groove is narrow and deep. Whether A-DNA occurs in cells is uncertain, but there is evidence for some short stretches (tracts) of Z-DNA in both bacteria and eukaryotes. These Z-DNA tracts may play a role (as yet undefined) in regulating the expression of some genes or in genetic recombination. Specific DNA Sequences Adopt Unusual Structures Other sequence-dependent structural variations found in larger chromosomes may affect the function and metabolism of the DNA segments in their immediate vicinity. For example, bends occur in the DNA helix, wherever four or more adenosine residues appear sequentially in one strand. Six adenosines in a row produce a bend of about 188. The bending observed with this and other sequences may be important in the binding of some proteins to DNA. A relatively common type of DNA sequence is a **palindrome**. A palindrome is a word, phrase, or sentence spelt identically and read forward or backwards; two examples are ROTATOR and NURSES RUN. The term is applied to DNA regions with **inverted** **repeats** of base sequence having twofold symmetry over two strands of DNA. Such sequences are self-complementary within each strand and, therefore, have the potential to form **hairpin** or **cruciform** (cross-shaped) structures. When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a **mirror repeat**. Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures. Sequences of these types are found in virtually every large DNA molecule and can encompass a few base pairs or thousands. The extent to which palindromes occur as cruciforms in cells is unknown, although some cruciform structures have been demonstrated in vivo in *Escherichia coli*. Self-complementary sequences cause isolated single strands of DNA (or RNA) in solution to fold into complex structures containing multiple hairpins. RNA is found in both the nucleus and the cytoplasm, and an increase in protein synthesis is accompanied by an increase in the amount of cytoplasmic RNA and an increase in its rate of turnover. These and other observations led several researchers to suggest that RNA carries genetic information from DNA to the protein biosynthetic machinery of the ribosome. In 1961, François Jacob and Jacques Monod presented a unified (and essentially correct) picture of many aspects of this process. They proposed the name \"messenger RNA\" (mRNA) for that portion of the total cellular RNA carrying the genetic information from DNA to the ribosomes, where the messengers provide the templates that specify amino acid sequences in polypeptide chains. Although mRNAs from different genes can vary significantly in length, the mRNAs from a particular gene generally have a defined size. The process of forming mRNA on a DNA template is known as **transcription**. A single mRNA molecule may code for one or several polypeptide chains in bacteria and archaea. If it carries the code for only one polypeptide, the mRNA is **monocistronic**; if it codes for two or more different polypeptides, the mRNA is **polycistronic**. In eukaryotes, most mRNAs are monocistronic. \"cistron\" here refers to a gene. the term itself has historical roots in the science of genetics, and its formal genetic definition is beyond the scope of this text.) The minimum length of an mRNA is set by the length of the polypeptide chain for which it codes. For example, a polypeptide chain of 100 amino acid residues requires an RNA coding sequence of at least 300 nucleotides because a nucleotide triplet codes each amino acid. However, mRNAs transcribed from DNA are always somewhat longer than the length needed to code for a polypeptide sequence (or sequences). The additional noncoding RNA includes sequences that regulate protein synthesis Tertiary structure of DNA Tertiary structure refers to the locations of the atoms in three-dimensional space, considering geometrical and thermic constraints. The two antiparallel polynucleotide chains of double-helical DNA are **complementary** to each other. Wherever adenine occurs in one chain, thymine is found in the other; Wherever guanine occurs in one chain, cytosine is found in the other. The hydrophilic backbones of alternating deoxyribose and phosphate groups are outside the double helix, facing the surrounding water. The furanose ring of each deoxyribose is in the C-2 endo conformation (depending on whether the atom is displaced to the same side of the plane as C-59 or to the opposite side). Both strands\' purine and pyrimidine bases are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis. The two strands create a major groove and a **minor groove**.  Nucleic acid quaternary structure It refers to the interactions between separate nucleic acid molecules or nucleic acid molecules and proteins (like histones and protamines) to form chromatin. Chromatin Chromatin is a complex of macromolecules found in eukaryotic cells, consisting of DNA, protein, and RNA. The primary functions of chromatin are: to package DNA into a smaller volume, to reinforce the DNA to allow mitosis, to prevent DNA damage, and to controlgene expressionand replication. Chromatin is organised on three basic levels: -primary (nucleosome) -secondary (solenoid) -tertiary/quaternary (final folding into chromosome shape) **Nucleic Acid Chemistry** The role of DNA as a repository of genetic information depends in part on its inherent stability. The chemical transformations that do occur are generally very slow in the absence of an enzyme catalyst. However, long-term information storage without alteration is so important to a cell that even very slow reactions that alter DNA structure can be physiologically significant. Processes such as carcinogenesis and ageing may be intimately linked to slowly accumulating, irreversible DNA alterations. Other non-destructive alterations also occur and are essential to function, such as the strand separation that must precede DNA replication or transcription. In addition to providing insights into physiological processes, our understanding of nucleic acid chemistry has given us a powerful array of technologies that have applications in molecular biology, medicine, and forensic science. **DNA denaturation: significance and factors** DNA is a remarkably flexible molecule. Considerable rotation is possible around several bonds in the sugar-phosphate backbone. Thermal fluctuation can produce bending, stretching, and unpairing(melting) of the strands. Solutions of carefully isolated, native DNA are highly viscous at pH 7.0 and room temperature (25 ℃). When such a solution is subjected to extremes of pH or temperatures above 80℃, its viscosity decreases sharply, indicating that the DNA has undergone a physical change. Heat and extreme pH cause denaturation, or melting, of double-helical DNA due to disruption of the hydrogen bonds between paired bases of the double helix to form unwound molecules. The covalent bonds in the DNA are NOT broken because they are strong. Slow cooling or neutralisation of pH causes renaturation of the denatured DNA molecule. i.e. the unwound segments of the two strands spontaneously rewind or anneal to yield the intact duplex. The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acid strands are paired. This is called the hypochromic effect. Denaturation of a double-stranded nucleic acid produces the opposite result: an increase in absorption called the hyperchromic effect. The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring UV absorption at 260 nm. Viral or bacterial DNA molecules in solution denature when they are heated slowly. Each species of DNA has a characteristic denaturation temperature, or melting point (*t*m; formally, the temperature at which half the DNA is present as separated single strands): the higher its content of GqC base pairs, the higher the melting point of the DNA. This is because GqC base pairs, with three hydrogen bonds, require more heat energy to dissociate than APT base pairs. Thus, the melting point of a DNA molecule, determined under fixed conditions of pH and ionic strength, can yield an estimate of its base composition. If denaturation conditions are carefully controlled, regions rich in APT base pairs will specifically denature while most of the DNA remains double-stranded. Such denatured regions (called bubbles) can be visualised with electron microscopy. Note that in the strand separation of DNA that occurs in vivo during processes such as DNA replication and transcription, The sites where these processes are initiated are often rich in APT base pairs, as we can see. Duplexes of two RNA strands or one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, RNA duplexes are more stable to heat denaturation than DNA duplexes. At neutral pH, denaturation of a double-helical RNA often requires temperatures 20 8C or higher than those required for denaturation of a DNA molecule with a comparable sequence, assuming the strands in each molecule are perfectly complementary. The stability of an RNA-DNA hybrid is generally intermediate between RNA and DNA duplexes. The physical basis for these differences in thermal stability is not known. Nucleotides and Nucleic Acids Undergo Nonenzymatic Transformations Purines and pyrimidines, along with the nucleotides of which they are a part, undergo spontaneous alterations in their covalent structure. The rate of these reactions is generally *very slow*, but they are physiologically significant because of the cell\'s very low tolerance for alterations in its genetic information. Alterations in DNA structure that produce permanent changes in the genetic information encoded therein are called **mutations**, and much evidence suggests an intimate link between the accumulation of mutations in an individual organism and the process of ageing and carcinogenesis. Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups (deamination). For example, cytosine deamination (in DNA) to uracil under typical cellular conditions occurs in about one of every 107 cytidine residues in 24 hours. This corresponds to about 100 spontaneous events per day, on average, in a mammalian cell. Deamination of adenine and guanine occurs at about 1/100th this rate. The slow cytosine deamination reaction seems innocuous enough, but it is almost certainly why DNA contains thymine rather than uracil. The product of cytosine deamination (uracil) is readily recognised as foreign in DNA and is removed by a repair system (Chapter 25). If DNA normally contained uracil, recognition of uracils resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent sequence changes as they were paired with adenines during replication. Cytosine deamination would gradually lead to a decrease in GqC base pairs and an increase in APU base pairs in the DNA of all cells. Over the millennia, cytosine deamination could eliminate GqC base pairs. Moreover, the genetic code depends on them. Establishing as one of the four bases in DNA, thymine may well have been one of the crucial turning points in evolution, making the long-term storage of genetic information possible. Another important reaction in deoxyribonucleotides is the hydrolysis of the *N*-*\_*-glycosyl bond between the base and the pentose to create a DNA lesion called an AP (apurinic, apyrimidinic) site or abasic site. This occurs at a higher rate for purines than for pyrimidines. As many as one in 105 purines (10,000 per mammalian cell) are lost from DNA every 24 hours under typical cellular conditions. Depurination of ribonucleotides and RNA is much slower and generally is not considered physiologically significant. In the test tube, the loss of purines can be accelerated by dilute acid. Incubation of DNA at pH 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid. Other reactions are promoted by radiation. UV light induces the condensation of two ethylene groups to form a cyclobutane ring. In the cell, the same reaction between adjacent pyrimidine bases in nucleic acids forms cyclobutane pyrimidine dimers. This happens most frequently between adjacent thymidine residues on the same DNA strand. A second type of pyrimidine dimer, a 6-4 photoproduct, is also formed during UV irradiation. Ionising radiation (x-rays and gamma rays) can cause ring opening and fragmentation of bases and breaks in the covalent backbone of nucleic acids. Virtually all life forms are exposed to energy radiation capable of causing chemical changes in DNA. Near-UV radiation (with wavelengths of 200 to 400 nm), which makes up a significant portion of the solar spectrum, is known to cause pyrimidine dimer formation and other chemical changes in the DNA of bacteria and human skin cells. We are subject to a constant field of ionising radiation in the form of cosmic rays, which can penetrate deep into the earth, as well as radiation emitted from radioactive elements, such as radium, plutonium, uranium, radon, 14C, and 3H. X-rays used in medical and dental examinations and radiation therapy for cancer and other diseases are another form of ionising radiation. It is estimated that UV and ionising radiations are responsible for about 10% of all DNA damage caused by environmental agents. DNA also may be damaged by reactive chemicals introduced into the environment as products of industrial activity. Such products may not be injurious per se. However, it may be metabolised by cells into forms that are. There are two prominent classes of such agents. \(1) deaminating agents, particularly nitrous acid (HNO~2~) or compounds that can be metabolised to nitrous acid or nitrites, and (2) alkylating agents. Nitrous acid, formed from organic precursors such as nitrosamines, nitrite and nitrate Salts are a potent accelerator for the deamination of bases. Bisulfite has similar effects. Both agents are used as preservatives in processed foods to prevent the growth of toxic bacteria. They do not seem to increase cancer risks significantly when used in this way, perhaps because they are used in small amounts and contribute only to the overall levels of DNA damage. (The potential health risk from food spoilage if these preservatives were not used is much greater.) Alkylating agents can alter certain bases of DNA. For example, the highly reactive chemical dimethylsulfate can methylate guanine to yield *O*6-methylguanine, which cannot base-pair with cytosine. Many similar reactions are brought about by alkylating agents normally present in cells, such as *S*-adenosyl methionine. The most important source of mutagenic alterations in DNA is oxidative damage. Excited-oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism. The hydroxyl radicals are responsible for most oxidative DNA damage of these species. Cells have an elaborate defence system to destroy reactive oxygen species, including catalase and superoxide dismutase enzymes that convert reactive oxygen species to harmless products. A fraction of these oxidants inevitably escape cellular defences, however, and DNA damage occurs through any of a large, complex group of reactions ranging from oxidation of deoxyribose and base moieties to strand breaks. Accurate estimates for the extent of this damage are not yet available, but every day, the DNA of each human cell is subjected to thousands of damaging oxidative reactions. This is merely a sampling of the best-understood reactions that damage DNA. Many carcinogenic compounds in food, water, or air exert their cancer-causing effects by modifying bases in DNA. Nevertheless, the integrity of DNA as a polymer is better maintained than RNA or protein because DNA is the only macromolecule that benefits from extensive biochemical repair systems. These repair processes greatly lessen the impact of DNA damage. Some Bases of DNA Are Methylated Certain nucleotide bases in DNA molecules are enzymatically methylated. Adenine and cytosine are methylated more often than guanine and thymine. Methylation is generally confined to specific sequences or regions of a DNA molecule. In some cases, the function of methylation is well understood; in others, the function remains unclear. All known DNA methylases use *S*-adenosylmethionine as a methyl group donor. E. coli has two prominent methylation systems. One serves as part of a defence mechanism that helps the cell to distinguish its DNA from foreign DNA by marking its DNA with methyl groups and destroying (foreign) DNA without the methyl groups (this is known as a restriction-modification system). The other system methylates adenosine residues within the sequence (59)GATC(39) to *N*6-methyladenosine. This is mediated by the Dam (*D*NA *a*denine *m*ethylation) methylase, a system component that repairs mismatched base pairs formed occasionally during DNA replication. In eukaryotic cells, about 5% of cytidine residues in DNA are methylated to 5-methylcytidine. Methylation is most common at CpG sequences, producing methyl-CpG symmetrically on both strands of the DNA. The extent of methylation of CpG sequences varies by molecular region in large eukaryotic DNA molecules. Nucleic acid hydrolysis The [nucleic acid](https://www.wikilectures.eu/index.php?title=Nucleic_acid&action=edit&redlink=1) chain can be cleaved either enzymatically or non-enzymatically. A 2\'-OH group is required for the alkaline hydrolysis of a polynucleotide, as its first step is the transfer of the bond from C5\' of the following nucleotide to C2\' of the same nucleotide. This breaks the axis of the polynucleotide and temporarily forms a cyclic 2\',3\'-phosphate, which is further degraded into a mixture of 2\' and 3\'-phosphates. It follows that heating in an alkaline environment hydrolyses only [RNA](https://www.wikilectures.eu/w/RNA), while [DNA](https://www.wikilectures.eu/w/DNA) is alkali resistant. By mild **acid hydrolysis** ([[pH]](https://www.wikilectures.eu/w/PH) about 3), the β-glycosidic bond of purine [nucleotide](https://www.wikilectures.eu/w/Nucleotide) is selectively cleaved. The result is a purine polynucleotide. The action of hydrazine can prepare a pyrimidine nucleic acid. RNA and DNA are split into components in a strongly acidic environment and at a higher temperature (HCl with a concentration of 6 mol.l−1, 175 °C). In addition, cytosine is deaminated to uracil, which must be considered when determining the base ratio in a polynucleotide. Enzymatic hydrolysis, catalysed by nucleases, is necessary to determine the primary sequence of nucleic acids. 3\'- and 5\'-**exonucleases** are distinguished, depending on which end of the polynucleotide they gradually cleave mononucleotides from. **Endonucleases** cleave bonds at a certain point within the chain, so the product tends to be oligonucleotides of unequal length. Endonucleases are pentose-specific; there are **ribonucleases (RNases)** and **deoxyribonucleases (DNases)**. Another criterion for the specificity of nucleases is the half of the phosphodiester bond they cleave---nucleases cleaving the bond closer to C3\' release nucleoside 3\'-phosphates. [**Restriction endonucleases**](https://www.wikilectures.eu/index.php?title=Restriction_endonucleases&action=edit&redlink=1) cleave the DNA double helix at a site of central symmetry in the nucleotide sequence. Snake venom phosphodiesterase is a 3\'-exonuclease cleaving α-bonds in both RNA and DNA. Bovine spleen phosphodiesterase is a 5\'-exonuclease cleaving polynucleotide b-bonds. Pancreatic RNase is an endonuclease that cleaves b-bonds in RNA if a pyrimidine nucleotide is attached to the a-bond of the given phosphate. RNase T cleaving the b-bonds of those phosphates where a purine nucleotide is attached to the a-bond was isolated from the fungus. REFERENCES **Chargaff, E.** (1950) Chemical specificity of nucleic acids and mechanism of their enzymic degradation. *Experientia* **6**, 201--209. **Chargaff, E.** (1951) Structure and function of nucleic acids as cell constituents. *Fed. Proc.* **10**, 654--659. ***Lehninger Principles of Biochemistry Fifth edition***