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Updated SQ 10-11.doc

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University of Waterloo
BIOL 308
Dragana Miskovic

STUDY QUESTIONS: LEC 10-11 1. List proteins (enzymes and other factors) involved in the process of DNA replication in E. coli. Explain briefly the role of each of these proteins in replication. − DnaB/helicase – move along dsDNA and separate strands (req ATP) − DnaA – initiates replication at OriC by recognizing four DnaA boxes (9bps) only if the DNA is negatively supercoiled. − DnaC -clamp ladder required by DnaB to escort it to DnaA forming the pre-priming complex − Single-stranded DNA binding proteins (SSB) – small proteins that bind to ssDNA cooperatively and prevent double helix from reforming and forced DNA to have extended conformation. − DnaG/primase/RNAP – synthesize RNA primers. Functions with DnaB to form next priming site (primosome) − DNA Pol II – involved in repair − DNA Pol III key enzyme that catalyzes synthesis of DNA from RNA primers − DNA Pol I – removes ribonucleotides including those directly linked to the DNA − RNase H – removes most the of the RNA primers except the last one that is directly linked to DNA − DNA ligase – links fragments on the lagging strand by forming phosphodiester bonds between 3’ OH and 5’ phosphate terminus of adjacent nts. − Topoisomerase – recognize and regulate supercoiling. 2. What is meant by replication being bidirectional? Semi- conservative? Continuous and discontinuous? Bidirectional  starts from a site of origin on the chromosomal material and replicates in both directions from that site due to DNA’s antiparallel nature– thus, replication bubble is formed, which expands. Semi-conservative  Helicase unwinds the dsDNA into two ssDNA, each of which is used as a template to synthesize the new DNA strand. Therefore, each new dsDNA molecule is 50% old and 50% newly replicated. Continuous in that on one strand, DNA is continually added off of one primer in the 5’ to 3’ direction. On the other strand, synthesis is discontinuous in that primers are continually added and replication occurs in the 5’ to 3’ direction until the previous strand is reached, when b-subunit releases core polymerase. 3. Contrast the role of DNA polymerase I and III in E. coli DNA replication. DNAPI removes rNTPs from primer and fills them in with dNTPs. Has 5’ to 3’ exonuclease activity, 5’ to 3’ polymerase activity, and 3’ to 5’ exonuclease activity to remove mismatches. Low processivity also. DNAPIII is highly processive and is absolutely required for DNA replication. It is a complex enzyme that adds dNTPs to both leading and lagging strands. It also has 3’ to 5’ exonuclease activity, but does not remove primers. It requires a primer to begin synthesizing DNA. 4. Which subunit of DNA polymerase III provides processivity? Which protein complex loads this subunit onto the DNA? The B-subunit provides processivity. The gamma-complex loads this subunit onto the RNA primer of DNA. 5. How can discontinuous synthesis of the lagging strand keep up with continuous synthesis of the leading strand? Primers are added and one of the core enzymes synthesizes DNA from it. A loop is formed because the core enzymes are attached together by the tau- subunits. Once the 135 bp or so fragment is synthesized, the core polymerase on the lagging strand is released, and binds upstream to another RNA primer. In this way, the lagging strand can keep up with the leading strand, which only runs off of one primer. 6. Why is decatenation required after replication of circular DNAs? After replication, the new daughter helices are linked together and are said to be catenated, but when they are in this form, part of the strands are yet to be replicated. Thus, the strands are denatured, then decatenated and this degrades the unreplicated terminus and finishes replication. 7. Why do eukaryotes need telomeres but prokaryotes do not? Prokaryotes have circular DNA, and thus there is always a 5’ end available for primers to be added to for DNA polymerase to bind to and synthesize the lagging strand. Eukaryotes do not have fixed ends as they are linear, and so there comes a point where a 3’ overhang will occur at the end of the chromosome, as RNA primase cannot add a primer right onto the exact end. 8. Thinking question: What would be the components necessary to make DNA in vitro by using DNA polymerase I? You would need dNTP, RNA primers, DNA of interest that contains target sequence you wish to copy, DNAPI 9. Thinking question: What properties would you expect an E. coli cell to have if it had a temperature – sensitive mutation in the gene for DNA ligase? At high temperatures, DNA ligase would not be able to ligate strands together. This will lead to failure of ligation of DNA fragments resulting in fragmented DNA that is possibly non functional. Therefore, if mitosis were to occur, the cell will die. 10. Thinking question: What properties would you expect an E. coli cell to have if it had a temperature – sensitive mutation in the gene for DNA polymerase I? The leading strand would be replicated normally however, the lagging strand would not be ab
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