DNA replication • DNA replication • Process of Replication in E. coli • Origin of replication • Role of Primase: RNA Primer • Elongation • Lagging strand synthesis: Okazaki fragments • Error rate of DNA synthesis • Eukaryotic Replication Types of replication Meselson-Stahl Experiment: Semi-conservative replication Eukaryotic chromosomes with base Analog 5-Bromodeoxyuridine with staining Process of Replication in E. coli Polymerases • DNA Polymerases • I,II, III • No initiation of replication • Primase: RNA polymerase • Lays down RNA nucleotides (primer) Origin of replication • Origin: 245 bp, containing repeats • Proteins involved, DNA A (initial denaturing), DNA B and C (further opening/destabilize helix) • unwinding of the helix: helicases (DNA B/C) • stabilization of the helix: single stranded binding proteins • role of topoisomerases, DNA gyrases Initiation Elongation • Anti-parallel strands • DNA Polymerase III • Leading strand synthesis DNA polymerase Replisome Lagging strand synthesis • Role of DNA Polymerase I • removal of primer • exonuclease activity • DNA ligase DNA Ligase Proofreading • Error rate of DNA synthesis • Proofreading • Base Pairing rules Eukayotic Replication • Multiple origins • Polymerases • Linear chromosomes Multiple origins Eukaryotic DNA Replication • DNA helicase promotes unwinding at the replication fork, • DNA pol δ with RFC and PCNA synthesizes DNA on the leading strand. • DNA pol α initiates synthesis on the lagging strand by generating an RNA primer (red segment) followed by a short segment of DNA. Then, RFC and PCNA load a second DNA polymerase (δ or ε ) to continue synthesis of the Okazaki fragment. • B, as DNA pol δ approaches the downstream Okazaki fragment, • Cleavage by RNase H1 removes the initiator RNA primer leaving a single 5 -ribonucleotide. Then, FEN1/RTH1 removes the 5 -ribonucleotide. The resulting nick is sealed by DNA ligase. Volume 272, Number 8, Issue of February 21, 1997 pp. 4647-4650 ©1997 by The American Society for Biochemistry and Molecular Biology, Inc. Linear chromosomes Telomerase= ribonucleoprotein with RNA, template for DNA synthesis.