Nucleic Acids and DNA

Nucleic Acids and Proteins

• Nucleic acids are key molecules in medical laboratories for handling and analysis.
• The nucleic acid analysis allows for the identification of normal and pathological traits.
• Analysis results in effective prevention and treatment of disease.

DNA Macromolecule

• Deoxyribonucleic acid (DNA) is a macromolecule composed of carbon, nitrogen, oxygen, phosphorous, and hydrogen.
• DNA assembles from nucleotides with a phosphorylated ribose sugar and a nitrogen base.
• DNA includes the four nitrogen bases adenine, cytosine, guanine, and thymine.
• Nitrogen bases attach to a deoxyribose sugar, to form a polymer with other nucleotides through a phosphodiester bond.
• Two DNA strands form the DNA double helix.
• DNA stores information based on the nucleotide sequence of its polymer

Discovery and makeup of DNA

• Johann Friedrich Miescher discovered DNA in 1869 via viscous substance from white blood cells.
• Miescher named the substance "nuclein," which had 14% nitrogen and 2.5% phosphorus.
• James Watson and Francis Crick described the double helix structure.
• Rosalind Franklin's diffraction analyses supported the findings of Watson and Crick.
• The helical DNA structure comes from physicochemical demands of the linear nucleotide arrangement.

Nucleotides

• Nucleotides have a molecular weight of about 700 kd.
• A nucleoside is a nitrogen base bound to an unphosphorylated sugar.
• Adenosine (A), guanosine (G), cytidine (C), and thymidine (T) are examples of nucleosides.
• A phosphorylated ribose sugar makes a molecule a nucleotide (nucleoside mono-, di-, or triphosphate).
• Free nucleotides include deoxyribonucleoside triphosphates like dATP, designated as A, C, G, and T.
• Nucleotides convert to nucleosides via hydrolysis.
• The five-carbon sugar of DNA is deoxyribose, which is ribose with a hydrogen atom linked to the number-two carbon, rather than a hydroxyl group.
• Hydroxyl group on the third carbon is vital for phosphodiester bond formation, establishing the DNA backbone.
• Nitrogen bases are planar carbon-nitrogen rings with amine and ketone substitutions and single/double ring bonds.
• Pyrimidines have a single-ring structure (thymine, cytosine).
• Purines have a double-ring structure (guanine, adenine).
• Nucleotide molecules are numbered by ring positions from the nitrogenous base
• Carbons on the ribose sugar are numbered 1' to 5', distinguishing from nitrogen base ring positions.

Helical Forms

• Original double helix by Watson and Crick becomes hydrated B-form DNA and it has 10.5 base pairs (bp) per turn.
• Dehydrated DNA version becomes the A-form with about 11 bp per turn.
• A and B-form DNA are right-handed helices.
• Stress and torsion can convert the double helix into a left-handed Z-form with altered sugar-base bonds.
• Z-DNA presents in areas of chromosomes under torsional stress from unwinding for transcription or metabolic functions.

Base Pairing

Two DNA strands form hydrogen bonds with each other in a specific way

• Guanines in one chain form three hydrogen bonds with cytosines in the opposite chain
• Adenines form two hydrogen bonds with thymines as well
• Single nucleic acid strands can bind or hybridize to single strands that have the corresponding bases
• Hydrogen bonds are key to specificity of most nucleic acid-based tests Specific hydrogen bond formation is how the information held in nucleotides is maintained
• DNA is polymerized, added nucleotides, and is hydrogen bonds with the complementary nucleotide on the parental strand (A:T, G:C)
• Parental DNA is replicated with no loss of the nucleotide order.

Variations & Practical Applications

Base pairs other than A:T and G:C or mismatches (e.g., A:C, G:T, A:A) can distort the DNA helix and disrupt sequence information Natural nucleic acid alternatives can hold on to same chemical properties of DNA (and RNA)

• Pentopyranosyl-(2′→4′) oligonucleotide systems exhibits stronger and more selective base pairing than DNA or RNA
• Protein nucleic acids, has carbon-nitrogen peptide backbone, replacing the sugar-phosphate backbone use as alternatives to DNA and RNA hybridization probes.
• Used as enzyme-resistant alternatives to RNA in antisense RNA therapies.

Modified Nucleotides

Modified bases result from damage to DNA or have specific functions relating to gene expression Modified nucleotides serve as primitive immune system for bacteria and viruses

• Distinguishes between the host or invaders (restriction-modification [RM] system)
• Host can target unmodified for degradation for own modifications recognition
• Modified nucleosides serve for clinical applications
• Anticancer drugs like 5-bromouridine (5BrdU) and cytosine arabinoside (cytarabine, ara-C) are a modified thymidine and cytosine

Nucleic Acids and their chains

Built by nucleotides attached phosphate and hydroxyl groups on their sugars

• Chain grows with 5' phosphate group connection of incoming nucleotide to the 3' hydroxyl group of final growing chain
• This phosphate gives chain polarity with 5' phosphate end and 3' hydroxyl end
• DNA as oriented in a 5' to 3' direction, and the nucleotides are read in order by convention

Sugar-phosphate Backbones

Arranged at certain distances from one another in the double helic Form regions in the helix called the major and minor grooves. Major and minor grooves are sites of interaction with proteins that bind specific nucleotide sequences in DNA (binding/recognition sites) The helix can be penetrated by intercalating agents, that slide transversely into the center of the helix Denaturing agents: formamide and urea, displace hydrogen bonds causing separation the strands

DNA Replication

DNA double helix has two versions of data saved in the form of order or sequence of nucleotides on chain. The sequence are complementary, not identical, with 5' to 3' arrangement and they form hydrogen bonds

• DNA hydrogen bonds produce hybridization Single strands of DNA with identical sequences will not hybridize with each other.

Enzyme activity in Replication

• Seminal role ensures maintenance in key actions that keeps nucleotides in DNA during new generations.
• Erwin Chargaff discovered amount of adenine in DNA corresponded to amount of thymine and cytosine to guanine as well
• Complementary strands separate to produce strands that are similar and serve to copy mechanism guides.
• New strand becomes the "elongate strand" by hydrogen bonding of "complementary" incoming nucleotide and nitrogen base during template elongation
• Duplicated helix will consist of one template strand and one newly synthesized strand.
• Semiconservative replication was demonstrated by Matthew Meselson and Franklin Stahl, using equilibrium density in cesium gradient Heavy DNA, prepared through growth, separates hybrid from 14 to 15 DNA separation, differentiation of semi conservative replication, and about half in heavy DNA are main differentiating factors. DNA undergoing active replication can be watched through use of forks. Both are 3-5 orientation and not consistent by copying strands

Enzymes and strands

Small pieces of DNA, or Okazaki fragments, helps explain how strands are copied in fork Parent helix strands copied in the same manner

• "Leading strand" jumps and copies backwards This type of jumping, discontinuous is known as "lagging strand." -3 Hydroxyl oxygen is made available for use Requires presence of other based, a provision that is held with polymerase from enzymes

Primer for DNA Synthesis

Primase = catalyzing the synthesis of short RNA primers that prime synthesis DNA. Replication occurs repeatedly

Replication complex

DNA replication has all proteins for double strands Helicase and primase activity occurs at each E. coli has very high throughput and speed for genome

Types of Polymerases

DNA Polymerase I (pol I) was the first enzyme that was shown to catalyze replication, Pol II and III were later characterized with Pol III main portion in bacterial activity. I and II for gaps and discontinuities in synthesized. Functions are a mutli-subunit holoenzyme in vitro, this can require initiation, protein regulation and termination, and additional supplements. The core enzymes are catalytic in some form and have polymerase activity for leading and stand for leading syntheses.