DNA Replication: Steps of Formation PDF

10 DNA REPLICATION I : STEPS OF FORMATION ILOs By the end of this lecture, students will be able to 1. Interpret the importance of DNA replication rules in preserving human body 2. Identify the requirements of DNA replication 3. Discuss process of DNA replication 4....

10 DNA REPLICATION I : STEPS OF FORMATION ILOs By the end of this lecture, students will be able to 1. Interpret the importance of DNA replication rules in preserving human body 2. Identify the requirements of DNA replication 3. Discuss process of DNA replication 4. Correlate the benefits of altering specific steps of DNA replication by drugs to control certain disease states What is meant by replication? Replication is the process whereby DNA makes a copy on itself each time the cell divides, in order to supply daughter cells with a typical copy of DNA present in parent cells. In Eukaryotes, DNA replication occurs in the nucleus during S-phase of interphase (interval between two cell divisions).(Refer to cell cycle lecture) Rules of DNA replication in eukaryotes 1. DNA replication is semiconservative: Each DNA strand serves as a template for synthesis of a new strand producing two double-stranded DNA molecules, each with one new strand and one old strand. 2. Replication begins at multiple origins and usually proceeds bidirectionally. Having multiple origins of replication provides a mechanism for rapidly replicating the great length of eukaryotic DNA molecules. 3. Replication exhibits polarity: DNA synthesis proceeds in a 5` 3` direction and is semi- discontinuous (What do you think this means?) 4. Replication is very accurate: replication proceeds with an extraordinary degree of fidelity. Identification of origins of Replication A human chromosome is tens to hundreds of millions of base pairs long. Logistically, this means that replication would be far too slow if it proceeded from only a single origin. In reality, replication starts at a number of different sites, termed origins of replication, spaced 30,000 to 300,000 base pairs apart. From each origin, a replication fork (the point at which DNA strands unwind) is formed, and proceeds in either direction until they meet, or until they reach the end of their chromosome. The number of active origins of replication is variable. At times when rapid 1  duplication of DNA is required (e.g., cell division of the early embryo), more origins of replication may be active. To identify the points in DNA strands where replication should start, certain proteins called origin recognition complex (ORC)proteins, scan the DNA strands for regions rich in Adenine and thymine complementary bases (Can you deduce the reason?). Once found, the proteins bind to those regions, tagging them as origins of replication. (Figure 1) Steps of DNA replication Step 1: DNA Unwinding To be copied, the DNA double helix must first be unwound and the strands separated by breaking the hydrogen bonds between the nitrogenous bases. The process is catalyzed by enzymes called DNA helicases (Molecular scissors). (Figure 1) To prevent unwound DNA double strands from rewinding back, certain proteins called single-stranded DNA binding proteins, bind to unwound DNA strands. Local unwinding can cause overwinding, or supercoiling, of DNA downstream, the thing that can prevent DNA unwinding downstream. This supercoiling is prevented by proteins called DNA topoisomerases, which create breaks between nucleotides, allow the DNA to uncoil, and then re-anneal the broken nucleotides. ⮚ Topoisomerase I makes single-stranded breaks. ⮚ Topoisomerase II makes double-stranded breaks. Unwinding of DNA, causes the formation of a structure called replication fork As a rule, DNA replication should proceed in the 5’>>3’ direction on the newly synthesized strand, and it should proceed AWAY from the origin of replication. Figure 1: Initiation of replication 2 Step 2: RNA Primer Synthesis DNA polymerases, the enzymes responsible for replication, cannot initiate synthesis of a new strand by linking free nucleotides together. In addition to a template, DNA polymerase requires a primer, a short piece of RNA with a free 3' hydroxyl, which the DNA polymerase can elongate. RNA primers are synthesized by an enzyme called RNA primase, which is a component of a DNA polymerase- α protein complex. The RNA primase synthesizes a short RNA primer (about 8-12 bp long) and the DNA polymerase-δ and polymerase-ε extend this primer by adding deoxynucleotides. Step 3: DNA Polymerization (Figure 2) The new daughter DNA strand is synthesized by adding nucleotides to the RNA primer. Nucleotides are joined together by phosphodiester bonds between the 3' hydroxyl group of the growing strand and the 5' phosphate of the next nucleotide. DNA polymerases are enzymes responsible for polymerization of newly added nucleotides. Newly added nucleotides are in the triphosphate form dGTP, dCTP, dATP, and dTTP. Energy for the formation of the phosphodiester bond comes from breaking the high- energy phosphate bonds on the nucleotide triphosphate. e.g. ATP >>>>>>AMP+ PPi, GTP>>>>>>GMP+ PPi…. etc Each newly added nucleotide should be complementary (i.e., G-C or A-T) to the corresponding nucleotide in the parental strand (template strand). GOLDEN RULE: Replication of new strands proceeds in the 5' >> 3' away from the origin of replication. On one strand, polymerization proceeds continuously away from the origin of replication. This is the leading strand. However, on the other strand, called the lagging strand, if replication is to proceed in the 5' > 3' direction, this means it will proceed towards the origin of replication which is against the rule. To overcome this, the daughter strand is synthesized in a discontinuous fashion in the form of short fragments of DNA that are approximately 150 base pairs long. These are called Okazaki fragments. Each fragment is synthesized in the 5’>3’ direction (i.e towards the origin of replication), however, as each new fragment is synthesized, the body perceives as if the synthesis of the new strand is proceeding away from the origin of replication. Okazaki fragments are later joined together through the action of DNA ligase. (Figure 2) Types of eukaryotic DNA polymerases DNA polymerase α: Priming and initial synthesis DNA polymerase β: DNA repair (refer to DNA damage and repair lecture) 3  DNA polymerase y: mtDNA replication DNA polymerase δ: Lagging strand synthesis, proof reading activity(Refer to DNA replication II lecture) DNA polymerase ε: Leading strand synthesis, proof reading activity Figure 2: Elongation of newly synthesized DNA strand and formation of leading and lagging strands Step 4: Degrading RNA Primers The RNA primers are removed by a 5' >>> 3' exonuclease. The resulting gap is filled in by a DNA polymerase. (Figure 3) Step 5: Ligation Ligation is the creation of phosphodiester bonds between individual DNA fragments so that the whole strand becomes one continuous strand. This is catalyzed by an enzyme called DNA ligase. (Figure 3) This process continues until the entire strand has been replicated to form two identical daughter strands. 4  Figure 3.Degradation of primers and ligation of strands Clinical implications: Different steps of DNA replication are target of action of many anticancer and antimicrobial agents: 1. Incorporate into DNA to interfere with chain elongation and induce defective ligation of fragments of newly synthesized DNA (especially in rapidly proliferating cells) in cancer cells by using anticancer; or in viruses by using antiviral; acyclovir against Herpes simplex virus. 2. Inhibit topoisomerase enzyme in cancer cells, by using the anticancer or in bacterial cells using the antibacterial agents; (Quinolone antibiotics). 5 11 DNA REPLICATION 2: PROTECTIVE MEASURES AGAINST ABNORMALITIES ILOs By the end of this lecture, students will be able to 1. Describe the proofreading mechanism during and after DNA replication 2. Describe the action of telomerase enzyme 3. Interpret at which points can abnormalities in DNA replication occur 4. Deduce health consequences of DNA replication errors 5. Appraise the benefits of targeting viral reverse transcriptase in the combating of some viral infections. As you have studied in your previous lecture, DNA replication ends with the formation of an exact copy of the parent DNA molecule. To ensure that the newly synthesized strand is protected against abnormalities, some measures are done such as proof reading and telomere capping. I) Proof reading (Figure 1) Proof reading is the process whereby continuous check on the newly inserted nucleotide in the growing DNA strand is being done. This process ensures the high degree of fidelity in DNA replication. Errors in base pairing occur during DNA synthesis. These mismatches would result in a mutation if they were not detected and corrected by proof reading. A base-pair mismatch occurs at a frequency of 1 per 10,000 nucleotides. DNA polymerases ε, δ and γ proof-read base-pair mismatches because any base pairs other than adenosine-thymine (AT) and guanine-cytosine (GC) create an irregularity in the shape of the DNA helix. Any irregularity in the helix results in the activation of a 3’ to 5’ exonuclease activity in both DNA polymerase enzymes. In other words, the polymerase backs up (3’ to 5’ direction), removes the incorrect nucleotide, and then reinserts the correct nucleotide. These mechanisms reduce the overall error rate to 1 mismatch per 1010 nucleotide  Defects in proofreading can lead to point mutation and trinucleotide repeat disorders (Refer to genetic variation and mutation lecture) 1  Figure 1. Process of proofreading II) Telomeres and Telomerase (Figure 2) Telomeres are complexes of DNA plus proteins (collectively known as shelterin) located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases (Cellular enzymes that break down nucleic acid) , and allow repair systems to distinguish a true end from a break in dsDNA. In humans, telomeres consists of several thousand tandem repeats of a noncoding hexameric sequence, AGGGTT. Telomeres are synthesized by telomerase enzyme. Telomerase: This enzyme complex contains a protein that acts as a reverse transcriptase enzyme (synthesizes DNA from RNA) in addition to a short RNA template, which is used to synthesize tandemly repeating six-base sequences (AGGGTT). This repeat is synthesized repeatedly and added to the end of the chromosome. Telomerase is active in embryonic cells, germ cells and cancer cells. Telomere shortening: Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5′-end of the lagging strand, there is no way to fill in the remaining gap with DNA. (figure 3) 2  Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells, embryonic cells, stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is due to the high activity of telomerase in those cells. Telomeres may thus be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres provides insight into the biology of normal aging, diseases of premature aging (the progerias), and cancer. (How?) Figure 2. Telomeres formation 3 Clinical Implication Reverse transcriptases As seen with telomerase, reverse transcriptases are RNA-directed DNA polymerases. Retroviruses, such as SARS-Cov 2 virus (responsible for COVID-19), human immunodeficiency virus (HIV, causes AIDS), and hepatitis C virus (causes inflammation and cancer of liver), carry their genome in the form of RNA molecules. They have a reverse transcriptase system which they use inside the human body to reversely transcribe the RNA to DNA and start replication inside the body, using the body’s own replication system. This activity can be targeted by drugs to inhibit their replication 4