VI. THE CENTRAL DOGMA OF MOLECULAR BIOLOGY A. DNA Replication Process con DNA Replication Unwinding proteins/enzymes (helicases) denature the helix at the origin of synthesis (replication origin/origin), creating the replication fork. On the other hand, single-stranded DNA binding proteins (SSBPs) stabilize the newly created single strands of denatured DNA. 2, The replication fork moves away from the origin in both directions, producing replication bubbles. The movement of the replication fork causes torsional stress/tension due to increased coiling of the helix occurring ahead of the fork. The tension created is diminished by the action of the topoisomerase DNA gyrase. This enzyme cuts DNA strands, allowing uncoiling to occur, and then reseals the DNA strands. 3. Initiation of DNA synthesis occurs concurrently on both template strands and involves an RNA primer synthesized under the direction of a unique RNA polymerase called primase. The resultant RNAs are complementary to their DNA templates. 4, DNA polymerase Ill polymerizes complementary DNA strands by simultaneously elongating the existing primer in the 5' — 3 direction. 5. As the replication fork moves away from the origin, synthesis is continuous on the leading strand, but is discontinuous on the lagging strand, producing short polynucleotides called ‘Okazaki fragments. The gaps between the Okazaki fragments are also called nicks. 6. RNA primers are subsequently removed, and the resulting gaps are filled with DNA under the direction of DNA polymerase I. The final gaps between the existing DNA and the replacement DNA are closed by DNA ligase. 7. Along the lagging strand, Okazaki fragments are joined by DNA ligase. 8. As synthesis proceeds along both the leading and lagging strands, proofreading by DNA polymerase III occurs. Finally, semi-conservative replication is achieved. DNA Repair > Inhumans, spontaneous damage to the DNA (mutations), is quite high. Approximately 10,000 different DNA sites is damaged in every human cell occur in a period of 24 hours. > Most of these damages are automatically repaired by various mechanisms: 1. As previously described, nicks are repaired by DNA ligase. 2. The loss of a base is repaired with the aid of AP endonuclease. 3. Ata site where a relatively large part (few base pairs) of the DNA strand is lost, repair is executed in various steps: ‘a. Cleavage of the damaged part of the DNA strand: Endonuclease b. Removal of the damaged portion: Exonuclease c._ Insertion of newly synthesized DNA strand: DNA polymerase d._ Sealing of the break: DNA ligase 4. Repair of DNA caused by UV light is achieved by the product of at least eight genes. Enzymes/Proteins Involved in DNA Replication and DNA Repair Enzyme/Protein Functionis Cleave the phosphodiester bond within a polynucleotide chain a easy ‘such as Deoxyribonuclease | which cuts DNA relatively Endonuclease i nonspectcaly Exonuclease 35" & 53" exonuclease activity DNA ligase Joins Okazaki fragments Removallrelieving of torsional stress/tension by breaking the DNA gyrase /topoisomerase | 00Uble strand ahead ofthe replication fork and making full rotation/s to unwind the helix. Once tension is removed, the broken strands of DNA are rejoined. DNA polymerase | Aids in filing gaps caused by removal of primersDNA polymerase Ill Synthesis of daughter strands; proofreading Helicase Unwinds the double helix, separating the parental strands ‘Single-stranded binding protein_| Stabilization of single strands of unwound DNA Primase /RNA polymerase _| Aids in the synthesis of the primer Terminologies Associated with DNA Replication Terms Lagging strand ‘Description New DNA strand that is synthesized in pieces/discontinuous Leading strand New DNA strand that is continuously synthesized Nicks ‘The spacesigaps between Okazaki fragments ‘Okazaki fragments The pieces of the lagging strand Replication bubbles Bubble-shaped structures formed from the origin as the replication occur in both directions Replication fork Region where parental strands are separating, and new strands are being synthesized, Replication origin Position along a DNA strand at which replication begins Primer/RNA primer First few nucleotides (up to 12) synthesized for elongation of a new daughter strand Nucleotide/RNA chain involved in the initiation of DNA synthesis Molecular Structure of a Gene > Genes are chemically composed of DNA and are situated in the chromosomes and are responsible for the determination of inheritable characters vvy Estimated that there are 30,000-35,000 genes located on 23 human chromosomes ‘Arranged in a single linear order in the chromosomes like the arrangement of beads on a string ‘Two kinds of genes in the chromosomes: 1. Structural genes — synthesis of specific proteins; a segment of DNA which contains the informationicode for synthesis of one complete polypeptide chain (or an enzyme) 2. ControvRegulatory genes ~ regulation of the activity of structural genes by either promoting or inhi a product 9 ling the sequences of events by which a structural gene is translated to Polypeptide chains are made of sequential arrangement of specific amino acids, so it was expected that there is contiguous sequence of DNA coding for these amino acids in a structural gene > Parts of a structural gene: 1. Exons ~ coding sequences 2. Inlrons ~ non-coding sequences interposed between exons 3. Flanking regions — important for gene regulation * Made up of DNA sequences which controls transcription * Called “promoter region’ and contains a “TATA” box (GGGCGGG sequences) and “CAT” box (CCAAT sequences) which are essential for transcription > (+) sense/codinginon-template strand vs. (-) antisense/non-coding/template strand B. Transcription > Genetic information stored in the DNA of a gene is transmitted to messenger RNA. > First step in the formation of a protein Process of Transcription 1. Transcription begins with the activation of transcription factors and the release of RNA polymerase in the promoter region of the gene, which causes breakage of hydrogen bonds. 2. Breakage of hydrogen bonds between nitrogenous base pairs causes the separation of the two strands of the DNA double helix to be separated from each other.3. RNA polymerase polymerizes complementary mRNA strands in the 5" - 3' direction, The produced nucleotide chain is known as a primary mRNA. A primary mRNA strand consists of all the sequences of a structural gene (ie. exons and introns.) 4. The non-coding sequences (introns) are then excised. The exons are spliced together to form a mature RNA which is relatively shorter in length. 5. Amethylguanine molecule attaches to the 5’ end and is known as the methyiguanine cap. This 5'cap protects the mRNA from degradation and facilitates mRNA transport to the cytoplasm. 6. On the other hand, the 3° end of mRNA is known as a poly (A) tail which, like the 5° cap, also protects mRNA from degradation and facilitates mRNA transport to the cytoplasm. 7. The mRNA migrates from the nucleus to the cytoplasm, then attaches to the ribosomes for protein synthesis. Alternative Splicing > Itwas thought that one gene produces only one protein or enzyme. The human genome only has approximately 30,000-35,000 genes, however, human proteins reach >100,000. > Alternative splicing is a process that causes one gene to give rise to more than one protein. > Exons are spliced in different patterns. The absence of one or tow exons in the mRNA changes the sequences of amino acids present in the resulting polypeptide chain. > Similarly, the function of the protein produced from the mRNA can also be modified by (a) phosphorylation, or, (b) combination with other proteins. > This explains why the number of proteins exceeds by almost three times the number of genes present in the human genome. C. Translation Components of the Translation System: 1. Messenger RNA — provides the coding sequence that determines the sequential arrangement of amino acids in the polypeptide chain 2. Ribosomal RNA/ribosome — consists of two subunits (small-40S and large-60S) which come together on the mRNA strand to form a mature ribosome. a. Small unit - reads the code on mRNA b. Large unit aligns successive RNA molecules and helps in the attachment of amino acids one by one via the peptide bonds 3. Transfer RNA — attached to a specific amino acid with the aid of the enzyme aminoacyl-tRNA synthetase. A tRNA attached to its amino acid is called a charged tRNA The Genetic Code Process of Translation 1. Initiation a. Initiation begins at 5' cap on the mRNA. Elongation factors (elF4, elF2, elF3 and elF5), 40S ribosome and an initiator RNA (which has UAC nucleotide sequence anticodon) forms the initiation complex. b. Once the initiation complex is formed, it moves along the mRNA in the 5' ~ 3° direction, Until the first AUG nucleotide sequence is encountered. c. Upon encountering the AUG nucleotide sequence, the 60S subunit attaches to the 40S subunit, releasing all initiation factors from the initiation complex except for the initiator tRNA. 4. The initiator tRNA along with its amino acid attaches to the AUG codon in the mRNA via hydrogen bonds. 2. Elongation a. At the start of elongation, the 40S subunit of ribosome moves one codon further on the mRNA, translating the codon present on it b. The new activated NA corresponding to the new codon is brought to the 60S subunit along with the corresponding amino acid.. The new amino acid joins the previous amino acid via peptide bond under the influence of peptidyl transferase in the 60S subunit. 4d. The 60S subunit then moves forward to catch up with the 40S subunit. e. After the formation of the peptide bond between the first two amino acids, one cycle of elongation is completed. The whole process is the repeated for the next codon. 3. Termination a. The stop codon is encountered on the mRNA strand, causing the binding of a release factor (RF) to the ribosome. The polypeptide chain gets detached from the tRNA to which itis attached. b. The RF is also release from the ribosome. . The 40S and the 60S subunit of the ribosome are separated from each other and recycled. D. Gene Regulation Gene regulation is the process of turning genes on and off. During early development, cells, begin to take on specific functions. Gene regulation ensures that the appropriate genes are expressed at the proper times. Gene regulation can also help an organism respond to its, environment. REFERENCES: Hatl, D. L. & Jones, E. W. (1998). Genetics principles and analysis, 4" ed. Sudbury, Massachusetts: Jones and Barlett Publishers. McPherson, R. A. & Pincus, M.R. (2011). Henry's clinical diagnosis and management by Laboratory Methods. 22" ed. Philadelphia, PA: Elsevier Saunders.