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lec.6 pgs 276-288 and 292-304.docx

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University of Toronto St. George

Fig. 5-19 A strand Directed Mismatch Repair system Removes Replication Errors That Escape from the Replication Machine: - Ecoli is capable of dividing once every 40 minutes, making it relatively easy to screen large populations to find a rare mutant cell. Mutator genes: genes that increase the rate of spontaneous mutations. - One such mutant makes a defective form of the 3'-5' proofreading exonuclease that is part of the DNA polymerase enzyme. Strand-directed mismatch repair: a system that detects the potential for distortion in the DNA Helix from the misfit between non-complementary base pairs. - If the proofreading system simply recognized a mismatch in newly replicated DNA and randomly corrected one of the two mismatched nucleotides, it would mistakenly "correct" the original template strand to match the error exactly half the time, thereby failing to lower the overall error rate. - To be effective, such a proofreading system must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred. - The strand-distinction mechanism used by the mismatch proofreading system in E. coli depends on the methylation of selected A residues in the DNA. - Methyl groups are added to all a residues in the sequence GATC, but not until sometime after the A has been incorporated into a newly synthesized DNA chain. - As a result, the only GAIC sequences that have not yet been methylated are in the new strands just behind a replication fork. The recognition of these unmethylated GATCs allows the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed. - The three-step process involves recognition of a mismatch, excision of the segment of DNA containing the mismatch from the newly synthesized strand, and resynthesized of the excised segment using the old strand as a template. - This strand-directed mismatch repair system reduces the number of errors made during DNA replication. - In eukaryotes, the mechanism for distinguishing the newly synthesized strand from the parental template strand at the site of a mismatch does not depend on DNA methylation. - Eukaryotes like yeast and the fruit fly have nicks in their newly synthesized lagging strand. - Nicks aka single strand breaks. DNA Topoisomerase prevents DNA tangling. - When a replication fork moves along double stranded dank it creates the “winding problem”. - For a replication fork to move the entire chromosome ahead must rotate rapidly. - This requires too much energy so a swivel is formed in the DNA helix by a protein called Topoisomerase. - Topoisomerase: A DNA Topoisomerase can be viewed as a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodeister bond in a DNA strand. This reaction is reversible, and the phosphodeister bond re-forms as the protein leaves. - There are two types of Topoisomerase type I and type II. - Type I: produces a transient single-strand break (or nick); this break in the phosphodeister backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodeister bond in the strand opposite the nick as a swivel point (Figure 5-22). Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. As a result, DNA replication can occur with the rotation of only a short length of helix-the part just ahead of the fork. - Type II: forms a covalent bond linkage to both strands of the DNA helix at the same time, making transient double -strand break in the helix. - These enzymes are activated by sites on chromosomes where two double helices cross over each other. - Once a Topoisomerase 2 bonds to the crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently. 1. It breaks one double helix reversibly to create a DNA gate 2. It causes the second, nearby double helix to pass through this break 3. It then reseals the break and dissociates from the DNA. - This way Topoisomerase 2 can efficiently separate 2 interlocked DNA circles. Same reaction also prevents severe DNA tangling problems that would arise during DNA replication. - P9.279 fig 5-22 DNA replication is similar in eukaryotes and bacteria. - There is more protein component in eukaryotic replication machines than there are in the bacterial analogs, even though the basic functions are the same. - Thus, for example, the eukaryotic single strand binding (SSB) protein is formed from three subunits, whereas only a single strand binding ssb protein is formed from three subunits, whereas only a single subunit is found in bacteria. - Similarly, the eukaryotic primase is incorporated into multi subunits enzymes that also contain DNA polymerase Called DNA polymerase alpha-primase. This protein complex begins each Okazaki fragment on the lagging strand - With RNA and then extends the RNA primer with a short length of DNA. the two main eukaryotic replicative polymerase 6 and r, come into play and complete each okazaki fragment while simultaneously extending the leading - Strand. - Nucleosome: A structural unit of a eukaryotic chromosome, consisting of a length of DNA coiled around a core of histones. - Also act as barriers that slow down the movement of DNA polymerase molecules, which may be why eukaryotic replication forks move only about one-tenth as fast as bacterial replication forks. - -DNA double helix is very stable; two strands are locked together by hydrogen bonds formed between the bases. - To use as a template double helix needs to be opened up. - The process of replication is begun but special "initiator proteins" that break the double strands apart by breaking hydrogen bonds. -Replication origin: the position is which DNA helix is first opened. - DNA rich in AT base pair bonds are easier to pull apart because they have fewer hydrogen bonds than CG base pairs. - Ecoli can only control DNA replication at initiation. Where it’s regulated. - Autoradiography: a technique used to determine the general pattern of eukaryotic chromosome replication. -it is done by adding 3H-thymidine, which is radioactive, and is then lysed and put on a glass that is coated with photographic emulsion. - To use this technique on an average sized human chromosome it will take 800 hours. - Experiments of this type however have shown that. (l) Replication origins tend to be activated in clusters, called replication units, of perhaps 20-80 origins. (2) New replication units seem to be activated at different times during the cell cycle until the entire DNA is replicated, a point that we return to below. (3) Within a replication unit, individual origins are spaced at intervals of 30,000-250,000 nucleotide pairs from one another. (4) As in bacteria, replication forks are formed in pairs and create a replication bubble as they move in opposite directions away from a common point of origin, stopping only when they collide head-on with a replication fork moving in the opposite direction (or when they reach a chromosome end). - In this way, many replication forks operate independently on each chromosome and yet form two complete daughter DNA helices. - DNA replication in most eukaryotic cells occurs only during a specific part of the cell division cycle, called the S phase. - Each chromosome has been replicated to produce 2 complete copies, which remain joined together at their centromeres until the M phase (mitosis). - Another approach for monitoring DNA replication i
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