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Molecular Events in DNA Replication

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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Molecular Events in DNA Replication

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  1. Molecular Events in DNA Replication

  2. 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

  3. 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

  4. 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

  5. DNA Replication – Initiation cont.

  6. 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

  7. 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

  8. Another look at a replication fork

  9. 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

  10. 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

  11. 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’

  12. 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

  13. 5’ 3’ Leading Strand DNA polymerase III 3’ SSBPs primase gyrase helicase 5’ 3’ 5’

  14. Lagging Strand Okazaki fragment

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

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