DNA REPLICATION

(b) Separation of strands Fig. 16-9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand Figure 16.9 A model for DNA replication: the basic concept

DNA Replication is semi-conservative: each replicated DNA molecule consists of one “old” and on “new” strand.

Getting Started Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” A eukaryotic chromosome may have hundreds or even thousands of origins of replication Replication proceeds in both directions from each origin, until the entire molecule is copied

Parental (template) strand Fig. 16-12a Origin of replication Parental (template) strand Daughter (new) strand Replication fork Double-stranded DNA molecule Replication bubble 0.5 µm Two daughter DNA molecules Figure 16.12 Origins of replication in E. coli and eukaryotes (a) Origins of replication in E. coli

Double-stranded DNA molecule Fig. 16-12b Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0.25 µm Bubble Replication fork Figure 16.12 Origins of replication in E. coli and eukaryotes Two daughter DNA molecules (b) Origins of replication in eukaryotes

1. Helicase breaks hydrogen bonds between bases, unzips and unwinds the helix

Helicase: Is an enzyme (a protein that speeds up chemical reactions) Is made during G1 Begins to unwind the DNA at the ORIGIN OF REPLICATION (a specific nucleotide sequence)

Helicase enzymes move in both directions from the Point of Origin, forming a REPLICATION BUBBLE. At either end of the replication bubble is a REPLICATION FORK, a Y-shaped region where the new strands of DNA are elongating.

2. Single stranded binding proteins hold the DNA strands apart Keeps the separated strands apart and stabilize the unwound DNA

RNA nucleotides bind with complementary base sequences under the direction of RNA primase. These RNA nucleotides act as a primer for DNA nucleotides. Primers are short segments of RNA, about 10 nucleotides long Must have a primer because DNA polymerase can only add nucleotides to another nucleotide

Primase

4. DNA polymerase III adds DNA nucleoside triphosphates to the RNA primer sequence in a 5’ to 3’ direction. Two of the phosphates are stripped off in the bonding

New nucleotides can only be added to the 3’ end of a growing DNA chain So we say DNA grows 5’ to 3’

Leading Strand: DNA polymerase III can synthesize a complementary strand on one side of the template in the 5’ to 3’ direction with no problem.

What about the other strand?? 5’ 3’ DNA Polymerase III Can only add to this side … AWAY from the replication fork

Lagging Strand DNA polymerase III must work away from the replication fork. Makes a short strand of DNA, called an Okazaki fragment. As the bubble widens, it can make another short strand, and so on.

RNA primers are removed and replaced with DNA nucleotides by DNA Polymerase I.

Along the lagging strand the Okazaki fragments are joined by DNA Ligase to form a single DNA strand.

Proofreading by DNA Polymerase III and I occurs, and replication is complete.

The Animation http://207.207.4.198/pub/flash/24/menu.swf

Animation: DNA Replication Fig. 16-13 Add onto the 3’ side (synthesized 5→3) Primase Single-strand binding proteins 3 Topoisomerase 5 3 RNA primer Figure 16.13 Some of the proteins involved in the initiation of DNA replication 5 5 3 Helicase Animation: DNA Replication

Overall directions of replication Fig. 16-15 Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 DNA poll III Parental DNA Figure 16.15 Synthesis of the leading strand during DNA replication 3 5 5 3 5

Overall directions of replication Fig. 16-16 Overview Origin of replication Leading strand Lagging strand Lagging strand 2 1 Leading strand Overall directions of replication 3 5 5 3 Template strand 3 RNA primer 3 5 1 5 Okazaki fragment 3 5 3 1 5 5 3 3 Figure 16.6 Synthesis of the lagging strand 2 1 5 3 5 3 5 2 1 5 3 3 1 5 2 Overall direction of replication

Table 16-1

Single-strand binding protein Overall directions of replication Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Single-strand binding protein Overall directions of replication Helicase Leading strand 5 DNA pol III 3 3 Primer Primase 5 Parental DNA 3 Figure 16.17 A summary of bacterial DNA replication DNA pol III Lagging strand 5 DNA pol I DNA ligase 4 3 5 3 2 1 3 5

DNA pol III synthesizes leading strand continuously Fig. 16-UN3 DNA pol III synthesizes leading strand continuously 3 5 Parental DNA DNA pol III starts DNA synthesis at 3 end of primer, continues in 5  3 direction 5 3 5 Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase Primase synthesizes a short RNA primer 3 5

Fig. 16-UN5

Accurate Only 1:1,000,000,000 nucleotides are incorrectly paired DNA replication is ... Accurate Only 1:1,000,000,000 nucleotides are incorrectly paired

DNA replication is ... Extremely rapid In prokaryotes, up to 500 nucleotides are added per second 50 per second in eukaryotes

Other Good Animations http://www.ncc.gmu.edu/dna/repanim.htm http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dna-rna2.swf http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html