3/23/2012. DNA Replication. DNA Replication. DNA Replication. Steps in DNA Replication. SBI4U1 Molecular Genetics

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1 SBI4U1 Molecular Genetics Recall: mitosis requires that each daughter cell has an exact copy of parent DNA. Ms. Ponvia The Watson-Crick model suggests how this occurs: Parent DNA molecule unzips, creating 2 single strands Each of these strands serves as a template for the creation of a complementary strand This way, the original template strand and the new complementary strand create 2 new identical DNA molecules This model is known as semiconservative, since each DNA molecule created is composed of: 1 original parent strand 1 newly created (daughter) strand Established in 1950 s by Meselson and Stahl Semi-conservative replication 1. Separating the DNA Strands DNA replication begins at specific sites known as origins of replication 1

2 A special enzyme called DNA helicase recognizes and binds to these sites, breaking the H-bonds This separated the two DNA strands To keep the strands apart, singlestranded binding proteins (SSBs) bind to the single strands and prevent the H-bonds from reforming (annealing) As the strands are separated, a Y-shaped region forms where the strands are still joined. On one strand, replication proceeds towards the fork, while on the other it proceeds away from the fork This forms a replication bubble: This is the replication fork 2. Building the Complementary Strands At the replication fork, an enzyme called DNA polymerase III (Pol III) is responsible for creating the new complementary DNA strands Pol III adds compounds called deoxyribonucleoside triphosphates (dntps) dntps are similar structure to ATP but for the sugar component a ribose in ATP and a deoxyribose in dntps: 2

3 As Pol III adds dntps to the growing DNA strand, they lose 2 phosphate groups This exergonic reaction provides the energy needed to create new nucleotides of DNA However, DNA Pol III only functions under certain conditions: Recall that DNA has polarity - one strand in the 3 to 5 direction, the other in the 5 to 3 direction DNA Pol III can only add dntps to the free 3 end of DNA, never to the 5 end. Thus, a new DNA strand can elongate ONLY in the 5 to 3 direction At the Replication Fork Along one strand, Pol III can create a continuous complementary strand by moving in the mandatory 5 to 3 direction This DNA strand is called the leading strand To elongate the other DNA strand, Pol III must work along the template away from the replication fork DNA made this way is called the lagging strand 3

4 The lagging strand is made in pieces called Okazaki fragments (Ofs) Another enzyme, DNA ligase, joins the Ofs into a single DNA strand Priming DNA Synthesis There is another restriction for Pol III: it cannot begin a DNA strand by joining the first nucleotides It requires an initial starting 3 end to start elongation This starting 3 end is provided by a series of bases called a primer, made of RNA, not DNA The enzyme primase builds the primer This way, Pol III adds the dntps to the 3 end of the primer using the template strand as a guide Only 1 primer is required for the leading strand. However for the lagging strand, each fragment must be primed. DNA polymerase I removes the RNA primers and replace it with DNA. DNA ligase then joins all gaps to form a continuous strand. Finally, Pol I and Pol III proofread the DNA and repair any mistakes by backtracking, removing & replacing incorrect nucleotides (ie. functioning as exonucleases) Corrections must be done immediately to avoid mistakes being copied in later replications. There are other methods as well that can fix errors after the completion of DNA replication. 4

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