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Lecture 3

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University of Toronto St. George
Melody Neumann

BIO130 – Week 3 DNA Replication, Repair, and Recombination Mutation Rates are Extremely Low • Occasional genetic changes enhance the long-term survival of a species, but the survival of an individual demands a high degree of genetic stability • The mutation rate is the rate at which observable changes occur in DNA sequences • The fibrinopeptides are able to function regardless of amino acid sequence o Sequence comparisons of the fibrinopeptides can therefore be used to estimate the mutation rate in the germ line, without worrying about missing mutations as a result of natural selection elimination o A typical protein of 400 amino acids will suffer an amino acid alteration roughly once every 200,000 years • Mutation rates for a single round of DNA replication tend to be roughly 1 nucleotide change per 10 nucleotides each time DNA is replicated Low Mutation Rates are Necessary for Life as we know it • Many mutations are deleterious, thus no species can afford to allow them to accumulate at a high rate in its germ cells • Although the observed mutation frequency is low, it is nevertheless thought to limit the number of essential proteins any organism can encode to perhaps 50, 000 • The cells of sexually reproducing organisms are of two types: o (1) germ cells, which transmit genetic information from parent to offspring o (2) somatic cells, which are the cells that form the body of the organism • Nucleotide changes in somatic cells can give rise to variant cells, such as those found in cancer o The diseases caused by these cells are due largely to an accumulation of changes in the DNA sequences of somatic cells o A significant increase in the mutation frequency would presumably cause a disastrous increase in the incidence of cancer by accelerating the rate at which somatic cell variants arise DNA REPLICATION MECHANISMS Base-Pairing Underlies DNA Replication and DNA Repair • DNA templating is the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence • DNA polymerase was the first nucleotide-polymerizing enzyme discovered BIO130 – Week 3 The DNA Replication Fork is Asymmetrical • During DNA replication inside a cell, each of the two original DNA strands serves as a template for the formation of an entire new strand • Since each new DNA double helix contains one original strand, and one new strand, the replication process is said to be “semiconservative” • In replication, there is a localized region of replication that moves progressively along the parental DNA double helix o Because of its Y-shaped structure, this active region is called a replication fork o At the replication fork, a multi-enzyme complex that contains the DNA polymerase synthesizes the DNA of both new daughter strands • Both daughter strands are created simultaneously, but DNA polymerase can read only in the 5’-to-3’ direction o Okazaki fragments are pieces of DNA that are polymerized in the 5’-to-3’ direction and joined together after their synthesis to create long DNA chains • A replication fork has an asymmetric structure o The DNA daughter strand that is synthesized continuously is known as the leading strands o The leading strand slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand  For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth  The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5’-to-3’ type of DNA polymerase The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms • Mispairing of nucleotides is possible between G and T, and rare tautomeric forms of the four DNA bases occur transiently, which can mispair without a change in helix geometry • Many “proofreading” mechanisms exist that act sequentially to correct any initial mispairings that might have occurred • DNA polymerase performs that first proofreading step just before a new nucleotide is added to the growing chain o Not only are the correct pairing more energetically favourable, before the nucleotide is covalently added to the growing chain, the DNA polymerase enzyme must undergo a conformational change in which its “fingers” tighten around the active site BIO130 – Week 3  This change occurs more readily with correct than incorrect pairings • The next error-correcting reaction is known as exonucleolytic proofreading, and it takes place immediately after those rare instances in which an incorrect nucleotide is covalently added to the growing chain o DNA polymerase absolutely require a previously formed base-paired 3’-OH end of a primer strand to continue elongating  DNA molecules with a mismatched nucleotide at the 3’-OH end of the primer strand are not effective as templates because the polymerase cannot extend such as strand  DNA polymerase molecules correct such a mismatched primer strand by means of a separate catalytic site. This 3’-to-5’ proofreading exonuclease clips off any unpaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3’-OH terminus that can prime DNA synthesis, (See page 270 figures 5-8 and 5-9 for clarification) • DNA polymerase requires a perfectly paired primer terminus. This requirement safeguards against errors • RNA polymerase doesn’t have the same requirement, and as a result experiences an error rate 100, 000 times greater than DNA polymerase A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand • For the leading strand, a special primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides • On the lagging side of the fork, every time the DNA polymerase completes a short DNA Okazaki fragment, it must start synthesizing a completely new fragment at a site further along the template strand o A special mechanism produces the base-paired primer strand required by the DNA polymerase molecule o The mechanism involves an enzyme called DNA primase, which uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand  RNA primer contains a properly base-paired nucleotide with a 3’-OH group at one end, it can be elongated by DNA polymerase at this end to begin an Okazaki fragment  The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5’ end of the previous fragment BIO130 – Week 3  To produce a continuous DNA chain from the many DNA fragments made on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA  An enzyme called DNA ligase then joins the 3’ end of the new DNA fragment to the 5’ end of the previous one to complete the process  Since a process that starts a chain anew cannot be efficient at self- correction, it makes sense to use RNA primers rather than DNA primers. If DNA primers were used, there wouldn’t be any need to go and check or replace them. RNA primers must be replaced, and to be replaced they must be checked for accuracy. Special Proteins Help to Open Up the DNA Double Helix in front of the Replication Fork • The DNA double helix is extremely stable under physiological conditions, and thus additional replication proteins are necessary to open the helix and provide the single- strand template • DNA helicases hydrolyze ATP when they are bound to single strands of DNA. o The hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to do mechanical work o DNA helicases use this principle to propel themselves rapidly along a DNA single strand  When they encounter a region of double helix, they continue to move along their strand, thereby
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