DNA Damage and Repair Quiz

• Nucleic acids are macromolecules, like proteins, carbohydrates, and lipids
• Nucleic acids 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 a nitrogenous base, 5-carbon sugar, and a phosphate group
• Nucleic acids carry the genetic and hereditary information needed to perform all life processes
• Storing and transmitting genetic information is fundamental for life

Structure of Nucleotides

• Nucleotides are the building blocks of nucleic acids
• Nucleotides contain a nitrogenous base, a pentose, and a phosphate
• A nucleoside is a molecule without the phosphate group

Nitrogenous Bases

• Purines have a two-ring structure and include Adenine (A) and Guanine (G)
• Pyrimidines have one ring and include Thymine (T), Cytosine (C), and Uracil (U)
• DNA contains Adenine, Guanine, Thymine, and Cytosine
• RNA contains Adenine, Guanine, Uracil, and Cytosine
• RNA contains ribose
• DNA contains deoxyribose

Nomenclature

• Nucleoside = sugar + base
• Nucleotide = sugar + base + phosphate

Roles of Functional Nucleotides

• Nucleotides are components of DNA and RNA
• The structure of every protein and cellular component is 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 enzyme cofactors and metabolic intermediates (ex., NAD+, FAD, FMN)

Nucleotide Derivatives

• NAD+/NADH
• NADP+/NADPH
• FAD/FADH2
• FMN/FMNH2
• c AMP, c GMP
• NAD+ & NADP+
• Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for many oxidoreductases, carrying 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, consisting 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
• 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 also a cofactor like FAD
• Cyclic adenosine monophosphate (cAMP) is an important second messenger in many biological processes
• cAMP is a derivative of adenosine triphosphate (ATP)
• The phosphate group attached to C5 forms a cyclic form with the –OH of C3
• 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
• cAMP binds to and regulates the function of some ion channels

DNA Primary Structure

• Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids
• Successive nucleotides of DNA and RNA are covalently linked through phosphate-group "bridges"
• The 5-phosphate hydroxyl group of one nucleotide unit joins to the 3 group of the next nucleotide, creating a phosphodiester linkage
• Backbones 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 or RNA strands have a 5' phosphate end and a 3' -OH end

DNA Secondary Structure

• The covalent backbones of nucleic acids consist of alternating phosphate and pentose residues
• The nitrogenous bases may be regarded as side groups joined to the backbone at regular intervals
• DNA is a double helix which 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 (covalent bond)
• Adenine (A) binds to Thymine (T) with two hydrogen bonds on the opposite strand
• Guanine (G) binds to Cytosine (C) with three hydrogen bonds on the opposite strand
• The two strands run in an antiparallel manner
• The DNA double helix, or duplex, is held together by hydrogen bonding between complementary base pairs and base-stacking interactions
• Base-stacking interactions contribute to the double helix's stability
• The number of purines (A+G) equals 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)
• The ratio of three bases can be calculated depending on the known ratio of any one base

DNA Tertiary Structure

• Tertiary structure refers to the locations of the atoms in three-dimensional space, considering geometrical and steric constraints
• Two antiparallel polynucleotide chains of double-helical DNA are complementary to each other
• Thymine is found where adenine occurs in one chain
• Cytosine is found wherever guanine occurs in one chain
• 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
• The B form of DNA described by Watson and Crick is right-handed
• One complete turn of the B-DNA double helix spans 34 Å
• One turn of B-DNA includes 10 base pairs
• Other forms of DNA include:
  • A-DNA is more compact than B-DNA
  • Z-DNA is left-handed and its bases are positioned more toward the periphery of the helix

Forms of DNA

• The plane of the base pairs in A-DNA is tilted about 20° relative to B-DNA base pairs
• A-DNA's base pairs are not perpendicular to the helix axis
• A-DNA structural changes deepen the major groove while making the minor groove shallower
• Z-form DNA has left-handed helical rotation
• Z-form DNA has12 base pairs per helical turn
• Z-form DNA appears more slender and elongated
• DNA backbone in Z-form takes on a zigzag appearance
• In Z-DNA The purine residues flip to the syn conformation, alternating with pyrimidines in the anti-conformation and the major groove is barely apparent
• Bends occur in the DNA helix, wherever four or more adenosine residues appear sequentially in one strand

Palindrome

• A palindrome is a word or phrase that is spelt identically read forward or backwards
• The term is applied to DNA regions with inverted repeats of base sequence having twofold symmetry over two strands of DNA
• Sequences are self-complementary within each strand and 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
• 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
• mRNA carries the genetic information from DNA to the ribosomes, where the messengers provide the templates that specify amino acid sequences in polypeptide chains
• Forming mRNA on a DNA template is transcription
• A single mRNA molecule may code for one or several polypeptide chains in bacteria and archaea
  • monocistronic: the mRNA carries the code for only one polypeptide
  • polycistronic: The mRNA codes for two or more different polypeptides
• In eukaryotes, most mRNAs are monocistronic
• The minimum length of an mRNA is set by the length of the polypeptide chain for which it codes

DNA Tertiary structure

• Tertiary structure considers the locations of the atoms in three-dimensional space, considering geometrical and thermic constraints
• Two antiparallel polynucleotide chains of double-helical DNA are complementary to each other
• Thymine is found in the other wherever adenine occurs in one chain
• Cytosine is found in the other wherever guanine occurs in one chain
• 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 refers to the interactions between separate nucleic acid molecules or nucleic acid molecules and proteins (like histones and protamines) to form chromatin
• Chromatin is a complex of DNA, protein, and RNA found in eukaryotic cells

Functions of Chromatin

• Package DNA into a smaller volume
• Reinforce the DNA to allow mitosis
• Prevent DNA damage
• Control gene expression and replication
• Chromatin is organised on three basic levels: primary (nucleosome), secondary (solenoid), tertiary/quaternary (final folding into chromosome shape)

Nucleic Acid Chemistry

• DNA's role as a repository of genetic information depends on its inherent stability
• 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

DNA Denaturation

• 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 °C)

• 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

• Slow cooling or neutralisation of pH causes renaturation of the denatured DNA molecule

• 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, called the hypochromic effect

• Denaturation of a double-stranded nucleic acid produces 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

• Each species of Viral or bacterial DNA has a characteristic denaturation temperature, or melting point (tm; 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

• GqC base pairs, with three hydrogen bonds, require more heat energy to dissociate than APT base pairs

• The melting point of a DNA molecule, determined under fixed conditions of pH and ionic strength, can yield an estimate of its base composition

• Duplexes of two RNA strands or one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured

• RNA duplexes are more stable to heat denaturation than DNA duplexes

• At neutral pH, denaturation of a double-helical RNA often requires temperatures 20 ºC or higher than those required for denaturation of a DNA molecule with a comparable sequence, assuming the strands in each molecule are perfectly complementary

• 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, called mutations

Alterations in DNA

• Cytosine deamination (in DNA) to uracil under typical cellular conditions occurs in about one of every 107 cytidine residues in 24 hours
• Deamination of adenine and guanine occurs at about 1/100th this rate
• DNA contains thymine rather than uracil so the product of cytosine deamination (uracil) is readily recognised as foreign in DNA and is removed by a repair system
• A DNA lesion called an AP (apurinic, apyrimidinic) site or abasic site occures by hydrolysis of the N-glycosyl bond between the base and the pentose
• UV light induces the condensation of two ethylene groups to form a cyclobutane ring between adjacent pyrimidine bases in nucleic acids forming cyclobutane pyrimidine dimers
• Ionising radiation (x-rays and gamma rays) can cause ring opening and fragmentation of bases and breaks in the covalent backbone of nucleic acids
• Near-UV radiation (with wavelengths of 200 to 400 nm) is known to cause pyrimidine dimer formation and other chemical changes in the DNA of bacteria and human skin cells
• Deaminating agents, particularly nitrous acid (HNO2), can damage DNA or bisulfite; alkylating agents can alter certain bases of DNA like the highly reactive chemical dimethylsulfate, and excited-oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals can cause mutagenic alterations in DNA
• Many carcinogenic compounds in food, water, or air exert their cancer-causing effects by modifying bases in DNA
• The integrity of DNA as a polymer is better maintained than RNA or protein

Methylation

• Certain nucleotide bases in DNA molecules are enzymatically methylated
• Adenine and cytosine are methylated more often than guanine and thymine
• All known DNA methylases use S-adenosylmethionine as a methyl group donor E. coli has two prominent methylation systems
• Eukaryotic cells methylate ~5% of cytidine residues in DNA to 5-methylcytidine
• Methylation is most common at CpG sequences, producing methyl-CpG symmetrically on both strands of the DNA

Nucleic acid hydrolysis

• The nucleic acid chain can be cleaved either enzymatically or non-enzymatically
• A 2'-OH group is required for the alkaline hydrolysis of a polynucleotide
• Heating in an alkaline environment hydrolyses only RNA, while DNA is alkali resistant
• By mild acid hydrolysis (pH about 3), the B-glycosidic bond of purine nucleotide is selectively cleaved
• Enzymatic hydrolysis, catalysed by nucleases, determines the primary sequence of nucleic acids
  • 3'- and 5'-exonucleases cleave mononucleotides from either end of the polynucleotide
  • Endonucleases cleave bonds at a certain point within the chain, so the product tends to be oligonucleotides of unequal length
  • Ribonucleases (RNases) and deoxyribonucleases (DNases) are pentose-specific
  • Restriction endonucleases cleave the DNA double helix at a site of central symmetry in the nucleotide sequence

Test your knowledge of DNA damage, repair mechanisms, and the chemical agents that can affect DNA structure. Questions cover topics such as pyrimidine dimer formation, base deamination, reactive oxygen species defense, alkylation, and base pairing.