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Molecular Events in DNA Replication. DNA Replication. DNA 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 Replication.
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DNA Replication • DNA 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 Replication • DNA 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 • DNA helicase – unwinds DNA to break its hydrogen bonds between complimentary bases • 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
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 gyraseand topoisomerase II – enzymes that loosens the tension in front of the replication fork
Replication Structures • Initiationcreatesan unwound, oval-shaped area called a replication bubble • Each Y-shaped region of the replication bubble is called areplication 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
Elongation Phase 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
Elongation • DNA 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
5’ 3’ Lagging Strand Elongation • Synthesis of lagging strand begins with the intialbinding of a short strand of RNA called an RNA primer to the parent strand • Primer is synthesized (made) by an enzyme called primase. 3’ 5’
Lagging Strand Elongation • 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 T A A
5’ 3’ Leading Strand DNA polymerase III 3’ SSBPs primase gyrase helicase 5’ 3’ 5’
Lagging Strand Okazaki fragment
Connecting Fragments on the Lagging Strands • DNA polymerase I – removes the RNA primer and fills gaps between fragments with DNA nucleotides • occurs in the 5’ 3’ direction • 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: • continuously on the leading strand • discontinuously on the lagging strand Replication Animation
Replication Overview - 1 • helicase 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 - 2 • two original parent strands serve as templates for the new daughter strands • daughter strands are produced in one of two methods • leading strand (continuous polymerization) • lagging strand (discrete polymerization) • 1000 – 2000 NT Okazaki fragments joined together
Replication Overview - 3 • primase 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