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1. DNA Replication Course Instructor Dr. Mahendra Ram Department of Chemical and Biochemical Engineering Email : mahendra.cbe @iitp.ac.in INDIAN INSTITUTE OF TECHNOLOGY PATNA 1

2. Revisiting DNA Structure DNA is a hereditary material in all living organisms. Located mainly in Nucleus in form of Chromatin . Double helix structure, two strands are complimentary and anti-parallel to each other. Genetic information in DNA is stored in form of sequence of four base pair (Adenine, Guanine, Cytosine, Thymine). Each DNA has ~ 3-4 billion base pairs. Base pairing: A=T (double hydrogen bond), G≡C (triple hydrogen bond) DNA is a poly- deoxyribonucleotide that contain many mono- deoxyribonucleotide covalently linked by 3 ′-5′ phosphodiester bond . 2

3. DNA structure 3

4. DNA Replication A process in which the two strands of the double helix unwind (unzip) and each acts as a template for the synthesis of a new strand of DNA . Well understood only for Prokaryotes ( Bacteria, namely E.coli). Central dogma of molecular biology (flow of genetic information) 4

5. DNA Replication (contd.) DNA replication is a basis of biological inheritance where identical copies of DNA are made before the cell division to transfer genetic information in daughter cells. This process synthesizes the daughter DNA from parent DNA by the enzyme DNA Polymerase . A semi-conservative process where parent strand serves as a template for a new strand and each of the two new synthesized DNA have one old and one new strand. DNA replication Parent DNA Daughter DNA 5

6. Characteristics of DNA Replication Semi-conservation (half of the parent DNA is conserved in each new double helix, paired with a newly synthesized complementary strand) Bi-directional (replication starts from unwinding the DNA at a particular point ( called origin of replication ), followed by the synthesis on each strand. The parent DNA and two newly formed DNA form a Y-shape structure called Replication fork ) Semi-continuous (leading strand is continuous while lagging strand is discontinuous) High fidelity (two new daughter strands are the exact copies of original DNA) 6

7. Semi-conservative DNA Replication The Meselson-Stahl experiment (1958) E-coli (bacteria) cells were grown for many generations in a medium (NH 4 Cl) contained only “heavy” isotope of nitrogen ( 15 N), so that all the nitrogen in their DNA was 15 N, as shown by a single band (blue) when centrifuged in a CsCl density gradient. ( b) Once the cells were transferred to a medium containing only light nitrogen ( 14 N) and allowed to grow until the cell population doubled. The DNA thus isolated formed single band (purple) at a higher position in CsCl . This indicated that DNA molecules of the daughter cells were hybrids containing one new 14 N strand and one parent 15 N strand. ( c) In another experiment, two DNA molecules: one consisting of two newly synthesized DNA strands and the other containing the two parent strands. Cells were allowed to double in 14 N medium. A second cycle of replication yielded a hybrid DNA band (purple) and another band (red ), containing only 14 N DNA , confirming semiconservative replication . 7

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9. Bi-directional DNA Replication 9 Replication bubble in Bacterial DNA

10. Replication Enzymes DNA Polymerase: These are family of enzymes, DNA Polymerase-I, II, III. DNA Polymerase-III extends the primers (elongation) by matching the correct nucleotides then joining adjacent nucleotides to each other . DNA Polymerase-II is involved in DNA repair and proof-reading. RNA primers are removed and replaced with DNA by DNA Polymerase-I. Helicase: Unwinds the DNA into two strands (breaks the hydrogen bond between base pairs) which act as template for synthesis of new complimentary strands. Primase: Provides an RNA primer (starter) to start polymerization . Ligase: The gaps between DNA fragments are sealed by DNA ligase. Topoisomerase: It works at the region ahead of the replication fork to prevent supercoiling. 10

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16. Steps in Replication (for bacteria E.coli) Separation of two complimentary strands ( replication begins at a point called origin of replication ). Formation of replication fork Y-shaped DNA helicase unwinds the double helix. Replication fork moves at the rate of 1000 nucleotides per sec. Single stranded binding (SSB) protein helps to keep the strands separated. Super-coiling problem ahead of replication fork interferes with the unwinding of double helix. DNA topoisomerase enzyme removes the supercoils (relieve torsional strain). These enzymes have both strand-cutting (nuclease) and strand-resealing (ligase) activities. 16

17. DNA strands at replication fork The leading strand is continuously synthesized in the 5 ′⟶ 3 ′ direction. The template is read in the opposite direction, 3 ′⟶ 5 ′ . The lagging strand is synthesized discontinuously in small DNA pieces ( Okazaki fragments ) in a direction opposite to that in which the replication fork moves. Each fragment requires a primer to start the synthesis. The Okazaki fragments are connected together by DNA ligase . In bacteria, Okazaki fragments are ~ 1,000 to 2,000 nucleotides long. In eukaryotic cells, they are ~150-200 nucleotides long . 17

18. Replication mechanism (contd.) Direction of replication DNA Polymerase is only able to read the parental nucleotide sequences in the 3 ′ ⟶ 5′ direction and they synthesize the new strand only in 5 ′ ⟶ 3 ′ (anti-parallel) direction. Synthesis of RNA primer An RNA primer is required for the initiation of synthesis of complimentary strand of DNA. RNA primer has a free –OH group at 3 ′ end of RNA strand. The enzyme Primase synthesizes the short stretches of RNA called primers (~10 nucleotides long) that are complimentary and anti-parallel to DNA template. These short RNA primers are constantly being synthesized at the replication fork on the lagging strand while only one RNA primer at the origin of replication is required on the leading strand. 18

19. Replication mechanism Chain elongation Elongation of new DNA strand by adding deoxy -ribonucleotides at 3 ′ end of growing chain. Elongation is catalyzed by DNA Polymerase-III enzyme. Removal of RNA primer and their replacement by DNA DNA Polymerase-I removes the RNA primer, replaces them with DNA and fills the gap between Okazaki fragments (stitching). DNA ligase action The final phosphodiester linkage (sealing) between the 5 ′ phosphate group and 3′ hydroxyl group on the chain is catalyzed by DNA Ligase. Termination Termination of replication by ter binding proteins at Ter sites by stopping the movement of DNA Polymerase-III and preventing the helicase from further unwinding of DNA. 19

20. DNA Proofreading and Repair DNA is copied by DNA Polymerase-III with very high accuracy. Incorrect nucleotide incorporation may lead to mutation . Errors are kept at very minimal levels by proofreading. Any mismatches are removed by 3 ′-5′ exonuclease activity of DNA Polymerase-II. 20

21. 21 Prokaryotic Replication Eukaryotic Replication It is a continuous process. This process occurs in the S-phase of cell cycle. Circular, double-stranded DNA Linear, double-stranded DNA with end The DNA replicates in the cytoplasm The DNA replicates in the nucleus Single origin of replication Multiple origins of replication Small amount of DNA The DNA is 50 times more than prokaryotic DNA DNA polymerase I and III are involved DNA polymerase ɑ, δ and ε are involved. Large okazaki fragments Small okazaki fragments The process is rapid, 2000 base pairs per second The process is slow, 100 base pairs per second Two circular chromosomes are obtained Two sister chromatids are obtained

22. Recombinant DNA Technology (Genetic Cloning) Transplantation of gene (small part of DNA) from one organism into another (host). Steps: Cleavage of DNA into specific fragments which can be manipulated. A new piece of DNA is then inserted into gap created by cleavage and then resealing the chain with DNA ligases and its introduction into host cells. When the recombination is successful, the transplanted gene synthesizes its designated protein. This technology is used in gene therapy where insulin drug is produced by E. coli bacteria (host) via recombinant DNA technology for the use by diabetic patients. 22

23. Insulin production via Recombinant DNA Technology Plasmids are circular DNA molecule of bacteria 23 (from Pancreas)