Nucleic Acid Chemistry PDF

Structure and function module (S1)-ZNU Part IV: Biochemistry Nucleic Acid Chemistry Definition: Nucleic acids are macromolecules contain the information for determining the amino acid sequence and hence the structure and function of all the proteins of a cell.Th...

Structure and function module (S1)-ZNU Part IV: Biochemistry Nucleic Acid Chemistry Definition: Nucleic acids are macromolecules contain the information for determining the amino acid sequence and hence the structure and function of all the proteins of a cell.They involve DNA and RNA. Structure of Nucleic Acids -They are polynucleotides which are formed of nucleotides linked by 3՛ to 5՛ phosphodiester bonds between the sugars. -Phosphodiester bond is formed between phosphate at 5՛ of one nucleotide and the OH of the 3՛ end of the pentose of adjacent nucleotide. -The pentoses and phosphates form the outer backbone (outer layer), while the bases form the inner layer. 302  Structure and function module (S1)-ZNU Part IV: Biochemistry Structure of nucleotides: -Nucleotide is the building unit of nucleic acid. -Nucleotide is composed of: A- nitrogenous base B-Pentose C-phosphate group A-Nitrogenous base -The nitrogen containing bases belong to two families: the purines and the pyrimidines. Purine bases include adenine (A) and Guanine (G). Both A and G are present in DNA and RNA. Pyrimidine Guanine bases include cytosine (C), uracil (U) and thymine (T). DNA contains T and C, whereas RNA contains U and C. T and U differ by only one methyl group, which is present on T but absent on U. 303 Structure and function module (S1)-ZNU Part IV: Biochemistry B-Pentose Sugar: There are two types of pentose sugar are found in nucleic acid: -Deoxyribose in DNA -Ribose in RNA The difference between the ribose and deoxy ribose lies on presence or absence of OH group at position 2 in sugar ring. C-Phosphate group It is attached to position 5 in sugar ring forming phosphodiester bond with the OH of the 3 end of the pentose of adjacent nucleotide. 304 Structure and function module (S1)-ZNU Part IV: Biochemistry Nucleosides Definition: Nucleoside is a molecule formed by association of nitrogenous base and pentose sugar. The sugar is ribose, a ribonucleoside is produced; if the sugar is 2-deoxyribose, a deoxyribonucleoside is produced. The ribonucleosides of A, G, C, and U are named adenosine, guanosine, cytidine, and uridine, respectively. The deoxyribonucleoside of A, G, C, and T have the added prefix, "deoxy-, for example deoxyadenosine (d adenosine). Deoxyribonucleic acid (DNA): - Deoxyribonucleic acid (DNA) contains all the information required to build the cells and tissues of an organism. - The information stored in DNA is arranged in hereditary units, now known as genes that control identifiable traits of an organism. 305 Structure and function module (S1)-ZNU Part IV: Biochemistry Structure of DNA: 1-Primary structure: - DNA is composed of a series of polymerized nucleotides, joined by phosphodiester bonds between the 5՛ and 3՛ carbons of deoxyribose units. - Purine bases are Adenine and Guanine. - Pyrimidine bases are Cytosine and Thiamine. - DNA is double-stranded, antiparallel. - The base on one strand determine the base sequence on the other strand, this is called complementary. 2-Secondary structure: - The two strands are joined by hydrogen bonding between their bases to form base-pairs. Adenine pairs with thymine, and guanine pairs with cytosine - Base pairing between deoxyadenosine and thymidine involves the formation of two hydrogen bonds. Three such bonds form between deoxycytidine and deoxyguanosine. - The two DNA strands run in opposite directions. One strand runs 5՛ to 3՛, and the other strand runs 3՛ to 5՛. The two DNA strands wind around each other, forming a double helix. - The bases are toward inside, while pentoses and phosphates are towards outside. 306 Structure and function module (S1)-ZNU Part IV: Biochemistry Organization of eukaryotic DNA: Chromosomal DNA is linear and is organized in loops and supercoiled by wrapping around small basic proteins called histones to form nucleosomes. 307 Structure and function module (S1)-ZNU Part IV: Biochemistry Nucleosome structure Nucleosomes are further organized into loops. The loops coil even further, forming a chromosome. This allows about 2 m of DNA in the human nucleus to form the 46 chromosomes with a total length of just 200μm. Histones (H1, H2A, H2B, H3, H4) Histones are involved in the packaging of DNA. Histone proteins are small globular proteins rich in arginine and lysine amino acids. They are positively charged which interact with negatively charged DNA. Ribonucleic acid RNA Primary structure: - Purine bases are adenine and guanine. - Pyrimidine bases are cytosine and uracil. - RNA is single stranded. Types of RNA A. mRNA: mRNA is formed in the nucleus from DNA by a process called transcription. It carries genetic information from DNA to ribosomes. Each 3 bases are called codon i.e. codes for a specific amino acid. B. tRNA tRNA molecules carry amino acids to ribosomes and ensure that they are incorporated into the appropriate positions in the growing polypeptide chain. For each amino acid , there are specific t-RNA. C. rRNA Ribosomes are subcellular ribonucleoprotein complexes on which protein synthesis occurs. Different types of ribosomes are found in prokaryotes and in the cytoplasm and mitochondria of eukaryotic cells. 308 Structure and function module (S1)-ZNU Part IV: Biochemistry 309 Structure and function module (S1)-ZNU Part IV: Biochemistry Molecular Biology DNA Replication - Definition: is the process in which the DNA within a cell makes an exact copy of itself. - In eukaryotes, this occurs during the S phase of the cell cycle. - DNA replication needs to occur before cell division (mitosis) so that each daughter cell has a complete set of hereditary material. - The process must be highly faithful. The characteristics of eukaryotic DNA synthesis 1-Semi conservative - DNA replication is said to be "semi-conservative" because each strand of the DNA double helix serves as a template for the synthesis of a new complementary DNA strand. - The newly replicated chromosome consists of one old and one new DNA strand 310 Structure and function module (S1)-ZNU Part IV: Biochemistry 2-Bi-directional with multiple origins of replication This means that replication begins at a site of origin and simultaneously moves out in both directions from this point. (1) Prokaryotes have one site of origin on each chromosome. (2) Eukaryotes have multiple sites of origin on each chromosome.  The requirements for DNA replication: 1-Template: Each strand of parent DNA serves as a template, upon which a new complementary strand will be formed. 2-Substrates (building units): Deoxyribonucleoside triphosphates are the building units for the synthesis of new strands of DNA. Four types must be present; dATP, dGTP, dCTP, dTTP. Anti-cancer drugs (e.g. methotrexate, 5-fluorouracil) stop cell division by interfering with dNTP supply, and dTTP. 3-Primer : - It is a short RNA strand about 10 - 20 nucleotides with free OH at 3' end. - This free OHs serves as an acceptor for nucleotides from DNApolymerases. - The RNA primer is synthesized by a specific RNA polymerase enzyme. (Primase). 311 Structure and function module (S1)-ZNU Part IV: Biochemistry 4-Major enzymes and other proteins involved in DNA replication I-DNA polymerases: - Chief enzyme mainly involved in replication and deoxynucleotide polymerization.These polymerases read the DNA template in 3' 5' direction and synthesize a new strand in 5' 3' direction. DNA polymerases have a proof-reading activity. - If a mismatch (uncomplimentary ) nucleotide is added, the enzyme removes it by its 3' 5' exonuclease activity. In prokaryotes: Three types of DNA polymerases found. They are DNA polymerase I, DNA polymerase II and DNA polymerase III. DNA polymerase I - Is primarily a repair enzyme and brings about deoxyribonucleotide polymerization. - It has both 5’→ 3’ and 3’ → 5’ exonuclease activities. Polymerase III - Is the main enzyme in the synthesis of both the leading and lagging strand. In eukaryotes: There is a large number of DNA polymerases, the major ones are:α, ε, β, γ, and δ. - DNA polymerase α is involved in generating primers for DNA replication. - DNA polymerase β is used exclusively for repair. - Polymerase δ acts as the lead polymerase in DNA synthesis. - Polymerase γ functions exclusively in mitochondria. II -DNA helicases: Required for the unwinding of (double-stranded) dsDNA. III-DNA Primase: Required for synthesis of the RNA primer. 312 Structure and function module (S1)-ZNU Part IV: Biochemistry IV- Nick sealing enzymes:Two enzymes: – Topoisomerases – DNA ligase. V- Single-strand binding proteins (SSB proteins).  Steps of DNA replication: 1-Separation of the two strands: - For the two strands of the parental double-helical DNA to be replicated, they must first separate (or "melt"). - They separate at least in a small region because the polymerases use only ssDNA (single-stranded DNA) as a template. In prokaryotic organisms - DNA replication begins at a single, unique nucleotide sequence, a site called the origin of replication(Ori). - This site includes a short sequence composed almost exclusively of AT base pairs. -In eukaryotes - Replication begins at multiple sites along the DNA helix to replicate rapidly the great length of the eukaryotic DNA molecules. - As the two strands unwind and separate they form a “V” where active synthesis occurs. - This region is called the replication fork. It moves along the DNA molecule as synthesis occurs. - Replication of dsDNA is bidirectional as the replication forks move in both directions away from the origin. 313 Structure and function module (S1)-ZNU Part IV: Biochemistry Origins of DNA replication (every ~150 kb) What are the proteins required for DNA strand separation? a - DNA protein: - It binds to specific nucleotide sequences at the origin of replication causing AT-rich regions in the origin to melt, and results in strand separation with the formation of localized regions of ssDNA. b- DNA helicases (Dna B): - These enzymes bind to ssDNA near the replication fork, and then move into the neighboring double-stranded region unwinding the double helix. - These enzymes use energy generated from ATP. 314 Structure and function module (S1)-ZNU Part IV: Biochemistry c-Single-stranded DNA-binding (SSB) proteins: - These proteins bind to the ssDNA generated by helicases. - They keep the two strands of DNA separated in the area of the replication origin, providing the single-stranded template required by polymerases, they protect the DNA from nucleases that cleave ssDNA. d- Topoisomerases: - Prevent the extreme supercoiling and super twisting of the parental helix that would result as a consequence of unwinding at a replication fork, which makes the separation more difficult and needs more energy. They are 2 types: Type I DNA topoisomerases - These enzymes reversibly cut one strand of the double helix. - They have both nuclease (strand-cutting) and ligase (strand-resealing) activities. - Each time a transient "nick" is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving ("relaxing") accumulated supercoils. Type II DNA topoisomerases (DNA Gyrase) - These enzymes bind tightly to the DNA double helix. - They make transient breaks in both strands leading to rapid relieve of supercoiled DNA. - It needs energy. 2- Formation of RNA primer: - DNA polymerases cannot initiate synthesis of a complementary strand of DNA without a primer. - It has a free hydroxyl group on the 3'-end of the RNA strand. 315 Structure and function module (S1)-ZNU Part IV: Biochemistry - This hydroxyl group serves as the first acceptor of a nucleotide by the action of DNA polymerase. Primase; RNA Polymerase (DnaG): - A specific RNA polymerase synthesizes the short stretches of RNA (approximately 10 nucleotides long) that are complementary and antiparallel to the DNA template. - It can synthesize primers using free NTPs as the substrate and the ssDNA as the template adds (ATP, UTP, CTP, GTP) one at a time to 5’  3’, anti- parallel to parent DNA strand. 3-Formation of two new strands. Both parent strands serve as a template for DNA synthesis producing two daughter strands. The DNA polymerases ((polymerase III in prokaryotes or DNA polymerase α for the first few nucleotides followed by DNA polymerase δ for the rest in eukaryotes)) are only able to “read” the template strand in the 3’ → 5’ direction, and they synthesize the new DNA strands in the antiparallel direction 5’→ 3’direction.Therefore, the two newly synthesized strands must grow in opposite directions—one in the 5’→ 3’ direction toward the replication fork (leading strand ) and one in the 5’→ 3’direction away from the replication fork (lagging strand). Using the OH of the 3' end of the RNA primer as acceptors of deoxyribonucleotides, DNA polymerases start biosynthesis of the new strands using dATP, dGTP, dCTP, dTTPThe selection of the new nucleotide is governed by proper base pairing rule. Leading strand: On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction toward the replication fork. This strand is referred to as the leading strand. Lagging strand: it is replicated in the opposite direction of the advancing replication fork in a discontinuous manner. It is synthesized in the form of small fragments termed Okazaki fragments; each is formed of a small DNA segment and connected to RNA primer. 316 Structure and function module (S1)-ZNU Part IV: Biochemistry 4-Excision of RNA primer and replacement by DNA. The RNA primers are removed by nucleases (e.g., RNase H in eukaryotes or polymerase I in prokaryotes) by 5’→ 3’exonuclease activity that can hydrolytically remove the RNA primer; then the resulting gaps are filled with the appropriate deoxyribonucleotides by another DNA polymerase (polymerase I in prokaryotes) in the 5’→ 3’ direction. Finally, the Okazaki fragments are joined by DNA ligase, an enzyme that catalyzes the formation of phosphodiester bonds between two polynucleotide chains. 317 Structure and function module (S1)-ZNU Part IV: Biochemistry Reverse transcriptase A reverse transcriptase is involved in the replication of retroviruses. Function : Following infection of a host cell, the viral enzyme, reverse transcriptase, uses the viral RNA as a template for the 5'- 3' synthesis of viral DNA, which then becomes integrated into host chromosomes and serve as a template for gene expression and from which new viral RNA genomes can be transcribed... Reverse transcriptases are called RNA dependent DNA polymerases. 318