DNA Replication Part 1 Principle Features

Figure 11.1 Identical base sequences Mechanistic Overview

Figure 11.2 More than 1 way to replicate a cat

Matthew Meselson and Franklin Stahl –Experimentally distinguished between daughter and parental strands How does it really happen?

11-9 Figure 11.3

11-10 DNA

Interpreting The Data After one generation, DNA is “half-heavy” consistent with both semi- conservative and dispersive models After ~ two generations, DNA is of two types: “light” and “half-heavy” consistent with only the semi- conservative model

DNA synthesis begins at a site termed the origin of replication –Each bacterial chromosome has only one Synthesis of DNA proceeds bidirectionally around the bacterial chromosome The replication forks eventually meet at the opposite side of the bacterial chromosome –This ends replication Bacterial DNA Replication

Figure 11.4 Theta Mode of Replication of Circular DNA

The origin of replication in E. coli is termed oriC –origin of Chromosomal replication Three types of DNA sequences in oriC are functionally significant –AT-rich region –DnaA boxes –GATC methylation sites Initiation of Replication

Figure 11.5 OriC of E. coli

Figure 11.6 Replication is initiated by binding of DnaA proteins to DnaA box sequences HU and IHF also bind causing the region to wrap around the DnaA proteins and melts the AT-rich region How the Ori Works: What happens at the Ori doesn’t stay at the Ori

Figure 11.6 Bidirectional replication Replication Initiation

Unwinding DNA DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them This generates positive supercoiling ahead of each replication fork –DNA gyrase travels ahead of the helicase and alleviates these supercoils Single-strand binding proteins bind to the separated DNA strands to keep them apart

DNA Synthesis is Bidirectional Two nascent, labeled strands at each fork means both parent strands serve as templates

Demonstration of Bi-Directional Synthesis

Nucleotide Polymerization Reaction

Implication of Bidirectional Synthesis RULE: Polymerization can only happen in 5'  3' direction Starting at 1 spot –only 1 strand can serve as template –but both strands do –therefore, one strand synthesized continuously leading strand –the other strand made discontinuously lagging stand

Okazaki Fragments If model is correct, should be able to find lagging strand fragments nt long DNA fragments Discovered by Reiji & Tuneko Okazaki Priming

Okazaki Fragments Begin with ~15nt RNA Primer

Origins, Replicons & Replisomes Origin of replication –1 per bacterial chromosome or plasmid –Many per eukaryotic chromosomes Replicon –Region of DNA replicated from a single origin –Consists of 2 replisomes moving in opposite directions Replisome –2 DNA pol holoenzymes and all associated proteins (primase, helicase, clamploader & clamps)

Figure Bidrectional DNA synthesis Replication forks will merge Multiple Origins of Replication

Eukaryotic Replicons In Action