Aashi Arora MCB 250 discussion WS Week 3 SP24

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Feb 20, 2024

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MCB 250 Spring 2024 Discussion Worksheet Week 3 – Feb. 8-12 1. a. Chromosomal DNA of both bacteria and eukaryotes is negatively supercoiled.  Why does negative supercoiling facilitate both replication and transcription of DNA?  It reduces torsional strain which makes it easier for the processes to proceed smoothly. b. In bacteria, Gyrase adds negative supercoils to DNA while other topoisomerases relax DNA.  These two reactions oppose each other.  Why do you think the cell is both adding and removing negative supercoils all the time? Adding and removing the negative supercoils in bacteria are essential in maintaining genomic stability, adapting to environmental changes, and regulating gene expression. 2. a. A closed circular DNA in its relaxed form has a linking number (Lk) of 500. A supercoiled form of this DNA has Lk = 495. This supercoiled DNA is treated with E. coli topoisomerase I and samples are withdrawn from the reaction at various times and electrophoresed in an agarose gel. Sketch how these gels would look. Include a lane with the relaxed form, a lane with the supercoiled DNA before the enzyme is added, and lanes at various times after the enzyme is added. The final lane should show what the gel looks like when the reaction has reached completion. The closed circular DNA in its relaxed form will migrate as a distinct band in the agarose gel. The supercoiled form will migrate faster due to its more compact structure. The final lane will show only the relaxed DNA band. b. Repeat the experiment using E. coli topoisomerase IV. What do you need in the reaction mix besides the enzyme to make it work? When the topo IV reaction is complete will the product be the same as for topo I? I would need Magnesium ions, along with ATP. The gel electrophoresis pattern will be similar to one with topoisomerase 1 showing a transition from 1
supercoiled to relaxed DNA. The final product may be different because topoisomerase resolves interlinked DNA molecules. c. Discuss the mechanism Topo I. Why is ATP not required to reform a phosphodiester bond after strand passage? Topoisomerase 1 acts by creating a transient single strand break in one of the DNA strands, allowing the passage of the other strand through the break before resealing. ATP isn’t required because the energy needed comes from the DNA itself. 3. In order to fit DNA into the nucleus of a eukaryotic cell it must be greatly compacted. To accomplish this, several levels of compaction are required. Discuss each of these levels beginning with the nucleosome. What proteins are required at each level and what structures are formed? The details of the lower level structures are much better understood than those of the higher level structures. Discuss. At what point in the cell division cycle is DNA most highly compacted and when is it least highly compacted? Nucleosomes are the fundamental organizational units of chromatin. DNA is bound tightly to beads for the proteins. The bead of each nucleosome contains eight histone molecules with two copies each of H2A, H2B, H3, and H4. Nucleosome cores appear to be organized into a structure called the 30 nm fiber which is a second level of organization. The third major class of chromatin proteins is the SMS proteins which in eukaryotes have two major types – cohesins and condensins. 4. The necessity to compact DNA presents a problem for the cell since DNA has to be accessible for replication (all of the DNA), transcription (some of the DNA), and repair (only damaged regions). Define heterochromatin and euchromatin in terms of accessibility and the level of compaction. How does the cell make DNA in highly compacted states available for these processes to occur? The coding region is called the euchromatin which is loosely bound in nature. The region undergoes rapid loosening and compaction cycles depending on the phase of the cell cycle. The non coding region is called the heterochromatin which is tightly bound in nature. The region is mostly compactly bound since it doesn’t go through transcription or translation. 2
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