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 baseAnalog 5-Bromodeoxyuridine withstaining

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 withRNA, template for DNA synthesis