YIJC [H2] CI2.2 DNA Replication (N)

YISHUN INNOVA JUNIOR COLLEGE 9744 H2 BIOLOGY LECTURE NOTES 1 CORE IDEA 2: GENETICS AND INHERITANCE TOPIC 2.2: DNA REPLICATION Learning Outcomes You should be able to: (b) describe the process of DNA replication and how the end replication problem arises Use the knowledge gained in this section in new situations or to solve related problems. References Reece, J B, Urry, L A, Cain, M L, Wasserman, S A, Minorsky, P V and Jackson, R B (201 7) Campbell Biology (11th Edition) (Pearson Higher Education) ISBN 0321739752 I. Introduction The structure of DNA was proposed by Watson and Crick in 195 3. With an understanding of DNA structure, experimental evidence supported the proposal that DNA replicates in a semi-conservative manner. During S phase of interphase, DNA content is doubled to form two sister chromatids joined at the centromere, in preparation of subsequent nuclear division. II. Evidence of Semi-Conservative DNA replication Matthew Meselson and Frank Stahl obtained evidence for the semi-conservative replication of DNA and published their results in 1958. The Meselson and Stahl experiment was as follows: ● Cultures of Escherichia coli (E. coli) bacteria were grown with the presence of ‘heavy’ isotope of nitrogen, 15N, instead of the ordinary ‘light’ isotope of nitrogen, 14N. ● After many generations, the bacteria culture all contained the heavy form, 15N. ● Some bacteria from the culture were treated to release their DNA into a solution. The solution with DNA was then centrifuged at high speed. ● The result was a single band of DNA, a heavy band that contained the 15N. This is known as the parental generation. H2

2 Fig. 2 Parental generation compared to control experiment with both 14N and 15N ● The remaining bacteria were placed in a 14N culture medium and allowed to grow for 20 minutes, which is the generation time for E. coli grown in optimal conditions. ● A sample of bacteria was then taken, processed, and centrifuged to produce the first generation (generation 1). ● This same process was continued so that the second and third generations were obtained, each being processed and centrifuged. Fig. 3 Results of second and third generations The Meselson and Stahl experimental results were as follows: • In the first generation, the DNA band occurred as an intermediate band in the middle of positions of 14N and 15N bands. This indicates that the DNA molecules (double helix) comprises of one 15N strand and one 14N strand. This shows that two 15N parental strands of DNA separated to act as a template for the replication of a new 14N daughter strand. After replication, the parental 15N strand rewinds with a newly synthesised 14N strand to form a new 14N15N daughter DNA molecule.

3 • In the second generation, DNA molecules from the first generation separated and each act as a template for the synthesis of a new 14N daughter strand. After replication, one 15N parental strand rewinds with newly synthesised 14N daughter strand to form a new daughter DNA molecule; while the other 14N parental strand rewinds with another newly synthesised 14N daughter strand to form a new daughter DNA molecule. Thus, one daughter DNA molecule consists of 14N15N forming an intermediate band while the 14N14N daughter molecule forms a light band. • This proceeds on with the third generation resulting in a larger proportion, thus thicker, light band (14N14N) • Thus, the experimental evidence supports semi-conservative mode of replication. Checkpoint 1 Illustrate the positions and thickness of the DNA bands in the fourth generation of E. coli cultured in 14N medium in Fig. 3.

4 III. Process of Semi-conservative Replication (i) Initiation • Helicase binds at the origin of replication (oriR). Helicase hydrolysed ATP to release energy to unwind and unzip the DNA double helix into two single strands by breaking the hydrogen bonds between the complementary base pairs. • This results in the formation of a replication bubble with 2 replication forks . Helicase continues to unwinds and separates the DNA bidirectionally away from the oriR. Fig. 4 Formation of replication bubble • Single strand binding proteins (SSBPs) bind to single -stranded regions of DNA near the replication fork to keep the strands apart to prevent the replication bubble from rewinding. • As replication continues, the unwinding of the double helix causes tighter twisting (supercoiling) in the DNA regions that are yet to unwind and unzip. • Topoisomerase / DNA gyrase helps relieve the supercoiling by breaking phosphodiester bonds in the sugar-phosphate backbone to allow the helix to untwist in the opposite direction before sealing the backbone. Fig. 5 Role of topoisomerase

5 Fig. 6 Helicase, single-strand binding proteins and topoisomerase at a replication fork (ii) Elongation • Primase binds to the separated DNA strands and synthesizes a short stretch of complementary RNA called a primer, using the DNA strand as a template, in the direction of 5’ → 3’. • It brings in ribonucleoside triphosphates (rNTPs) complementary to the DNA template and catalyses phosphodiester bonds between adjacent ribonucleotides. • The RNA primer provides a free 3’OH end for DNA polymerase to recognise and bind. Fig. 7 Role of primase The main DNA polymerase enzyme responsible for replication in prokaryotes is DNA polymerase III whereas in eukaryote, it is DNA polymerase δ. • DNA polymerase brings in free deoxyribonucleoside triphosphates (dNTPs) that are complementary to the parent DNA template. • DNA polymerase hydrolyses phosphate bonds in the 5' phosphate groups of incoming dNTPs to release energy for the synthesis of the daughter strand.

6 • DNA polymerase adds the resultant deoxyribonucleotides to the free 3’-OH end of the (RNA) primer and catalyses the formation of phosphodiester bonds between adjacent nucleotides. • Hence, the synthesis of the complementary daughter strand occurs in the 5’ → 3’ direction and the parental strand is read in the direction of 3’ → 5’. Fig. 8 Elongation at 3' OH end of the growing daughter strand during DNA replication in a bacterial (prokaryote) cell.

7 • Daughter strands that are synthesized continuously towards the replication fork in the direction of 5’  3’ using only one RNA primer are called leading strands. Fig. 9 Leading strand synthesis in prokaryotes • Daughter strands that are synthesized discontinuously away from the replication fork in the direction of 5’  3’ using multiple RNA primers are called lagging strands. • This is because the replication fork opens behind the 5' end of the lagging strand, thus a new primer needs to be synthesized each time a new polynucleotide strand is synthesized. • The lagging strand is thus synthesized discontinuous in the form of Okazaki fragments. (Right) Fig. 10 Lagging strand synthesis in prokaryotes

8 The daughter strands are synthesized in such a way because: • DNA polymerase cannot initiation the synthesis of a new DNA polynucleotide strand, • DNA polymerase can only elongate existing polynucleotide strand at the free 3'-OH end, thus it can only synthesize polynucleotide strand in the 5'  3' direction and, • pairing of DNA strands are in anti-parallel manner. • As DNA polymerase moves along the template, part of the enzyme proofreads the previous region. This is to check if proper base pairing has taken place between the bases. • If an incorrect deoxyribonucleotide was added, it would be removed by the 3’ → 5’ exonuclease activity of DNA polymerase a nd replaced with the correct deoxyribonucleotide. (Left) Fig. 11 Proofreading by DNA polymerase • The lagging strand is synthesized in Okazaki fragments, interspe