Molecular Events in DNA Replication

DNA ReplicationDNA replication is semi-conservative

= the mechanism of DNA replication in which each newly synthesized DNA molecule is composed of one strand from the original DNA molecule and one new strand

DNA ReplicationDNA replication starts at a number of sites along a

chromosome in eukaryotic cells – these are called the replication origin

In prokaryotic cells – generally only one site of replication

Complicated process – with dozens of proteins are involved and consists of 3 stages – initiation, elongation, and termination

Initiation – occurs at the origin of replication

1. DNA helicase – unwinds DNA to break its hydrogen bonds between complimentary bases

2. single-strand-binding proteins (SSBs) – bind to each of the single stranded pieces of DNA, stabilizing it

if there were nothing to keep the strands apart, they would reanneal (stick back together)

DNA Replication Animation

DNA Replication – Initiation cont.

Other Proteins / Enzymes of DNA Replication (Initiation stage)

as DNA is being unwound by helicase, DNA in front of the helicase gets bunched up

DNA gyrase and topoisomerase II – enzymes that loosens the tension in front of the replication fork

Replication Structures

Initiation creates an unwound, oval-shaped area called a replication bubble

Each Y-shaped region of the replication bubble is called a replication fork

Replication occurs in both directions from the origin of replication and as it proceeds, each replication fork moves in opposite directions along the DNA

Another look at a replication fork

ElongationPhase that synthesizes new DNA strands by

joining free nucleotides together

DNA polymerase III – enzyme which synthesizes nucleotide chains Adds nucleotides to the 3’ end of a growing

polynucleuotide strand in a continuous fashion towards the replication fork

The new strand generated from the parent strand that goes in the 5’ to 3’ direction is called the leading strand

ElongationDNA polymerase III – also synthesizes new DNA

from the parent strand that does not have a free 3’ hydroxyl end

This new strand is called the lagging strand

It is replicated away from the replication fork, in short segments and in a discontinuous manner

Lagging Strand Elongation1. Synthesis of lagging strand begins with the intial

binding of a short strand of RNA called an RNA primer to the parent strand

2. Primer is synthesized (made) by an enzyme called primase.

5’3’

5’ 3’

A

Lagging Strand Elongation3. With primer in place, DNA polymerase III

extends the strand by adding new nucleotides to the free 3’ hydroxyl end

These fragments extend away from the replicating forks and have to be repeated

Fragments called Okazaki fragments

AT

Leading Strand

5’

5’

3’

3’

helicase

SSBPs

gyrase

primase

5’

3’

DNA polymerase III

Lagging Strand

Okazaki fragment

Connecting Fragments on the Lagging Strands4. DNA polymerase I – removes the RNA primer and

fills gaps between fragments with DNA nucleotidesoccurs in the 5’ 3’ direction

5. DNA ligase – catalyses the joining of Okazaki fragments

How? connects the sugar-phosphate backbone of Okazaki fragments

Okazaki fragments are typically 1000 to 2000 nucleotides (NTs) in length

DNA Replication

Termination phase – occurs upon completion of the new DNA strands, and the two new DNA molecules separate from one another

Overview:DNA replication occurs:1. continuously on the leading strand2. discontinuously on the lagging strand

Replication Animation

Replication Overview - 1helicase unwinds the double stranded DNA structure

creating a replication fork

the single stranded region of the replication fork are maintained by SSBPs

gyrase relieves the tension ahead of the replication fork

Replication Overview - 2two original parent strands serve as templates for

the new daughter strands

daughter strands are produced in one of two methodsleading strand (continuous polymerization)lagging strand (discrete polymerization)

1000 – 2000 NT Okazaki fragments joined together

Replication Overview - 3primase begins each new daughter strand with a

short RNA primer

DNA polymerase III extends a DNA strand from the RNA primer

DNA polymerase I removes the RNA primer AND fills it in with DNA

DNA ligase joins the sugar-phosphate backbones of all adjacent DNA segments