DNA Replication Lecture (PDF)

Requirements for DNA Replication Nucleotides (dATP, dCTP, dGTP, dTTP) DNA template (Both strands of DNA) RNA primer (To initiate DNA synthesis) Enzymes : Helicases, primase, DNA polymerase, topoisomerase (gyrase) and ligase. Proteins : (i) Ori proteins (DnaA, DnaB & DnaC) (ii) Single-strand binding...

Requirements for DNA Replication Nucleotides (dATP, dCTP, dGTP, dTTP) DNA template (Both strands of DNA) RNA primer (To initiate DNA synthesis) Enzymes : Helicases, primase, DNA polymerase, topoisomerase (gyrase) and ligase. Proteins : (i) Ori proteins (DnaA, DnaB & DnaC) (ii) Single-strand binding protein (SSBP) 22 NOV 2023 MIBS BATCH 29 13 Important terms origin of replication (Ori/OriC) a particular sequence in a genome at which replication is initiated. leading strand the strand of DNA synthesized using the 3’-5’ DNA sequence as template. lagging strand the strand of DNA synthesized using the 5’-3’ DNA sequence as template. Replicon (replication unit) DNA replicated from a single origin. 22 NOV 2023 MIBS BATCH 29 14 Steps involved in DNA replication The 3 basic steps in DNA replication are: 1. Initiation Involves attachment of initiator proteins to oriC, unwinding of DNA by helicase and synthesis of RNA primer to initiate replication. 2. Elongation Involves elongation of new DNA strands with the aid of DNA polymerases. 3. Termination DNA replication is terminated. 22 NOV 2023 MIBS BATCH 29 15 Initiation Replication starts when DNA at the origin of replication (OriC) denatures to expose the bases, creating a replication fork. E. coli has one oriC. It is a AT rich sequence of about 245 bp required for initiation. In prokaryotes→ Single Origin of replication In eukaryotes → Multiple site of replication 3 major steps are involved in the initiation of DNA replication. 22 NOV 2023 MIBS BATCH 29 16 Initiation Initiation 3 major steps: Step 1: Attachment of initiator proteins to DNA molecule. ➢ The initiator protein (DnaA in E. coli) bind to the oriC. ➢ DNA helicase then unwinds the oriC & extends the single-stranded (ss) region for copying. Step 2: Binding of Single Strand Binding Protein (SSBP) & DNA unwinding ➢ SSBP binds to the ss region to protect it from breakage & prevent renaturation. ➢ As the DNA is unwound by DNA helicases & SSBP with some ATP, the resulting positive supercoiling (torsional stress) is relieved by topoisomerase by inducing transient single stranded breaks. Step 3: RNA Primer synthesis ➢ DNA primase binds helicase to form a primosome & synthesizes a short (2-10 nt) RNA primer to initiate the synthesis of the leading strand of the first replication fork. 22 NOV 2023 MIBS BATCH 29 17 Initiation helicase Initiator & single-stranded binding proteins (SSBP) replication fork In prokaryotes, only 2 replication forks are formed. Multiple replication forks are formed in eukaryotes. 22 NOV 2023 MIBS BATCH 29 Replication forks in prokaryotes. 18 Initiation: Formation of a replication bubble at the Ori and the initiation of the new DNA strand & SSBP 22 NOV 2023 Since multiple ORIs are present in eukaryotes, multiple replication bubbles are present as well. MIBS BATCH 29 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 19 Elongation Involves the synthesis of 2 strands of DNA using the parental DNA strands as template with the aid of DNA polymerases and ligase. The 2 strands of DNA are antiparallel (they run in opposite direction). From the initiation site [ORI], 1 parental strand runs in a 5’ - 3’, while the other strand is running in the 3’- 5’ direction. However, DNA synthesis is always in the 5’ - 3’ direction. Due to this 5’ - 3’ polarity and antiparallel nature of DNA, 2 different strands are formed. (1) Leading strand (2) Lagging strand 22 NOV 2023 MIBS BATCH 29 20 Elongation: Leading & Lagging strands - Leading strand uses 3’ - 5’ parent strand as the template. synthesis is continuous in 5’ – 3’ direction (towards replication fork). Only 1 RNA primer is present. Lagging strand uses 5’ - 3’ parent strand as the template. Synthesis is in fragments called the OKAZAKI fragments. synthesis is discontinuous in 5’ – 3’ direction (away from replication fork). Several RNA primers are present. 22 NOV 2023 In prokaryotes, the okazaki fragments are large (1000-2000 bases), while in eukaryotes, the fragments are shorter (100-200 bases) MIBS BATCH 29 21 Replication fork, leading & lagging strands RNA primer 22 NOV 2023 MIBS BATCH 29 22 Elongation Elongation involves 3 major steps: Step 1: DNA polymerases (Pol) extends the RNA primer. In prokaryotes, DNA polymerase (Pol) III extends the RNA pimer made by primase. In eukaryotes, DNA Pol α starts the elongation process. After addition of 20 nucleotides, elongation is taken over by Pol ε (on the leading strand) and Pol δ (on the lagging strand). Step 2: Removal of RNA primers & gap filling After DNA synthesis, DNA Pol I (in prokaryotes) and fills the gaps with new nucleotides. In eukaryotes, exonucleases remove the RNA primer & the gap is filled by polymerase δ. Step 3: Joining DNA fragments Ligase joins the ends of newly synthesized DNA fragments together. 22 NOV 2023 MIBS BATCH 29 23