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Chem 465 Biochemistry II Test 2 Spring 2018

Multiple choice (4 points apiece): 1. Bacterial : A) are always covalently joined to the bacterial chromosome. B) are composed of RNA. C) are never circular. D) cannot replicate when cells divide. E) often encode not normally essential to the bacterium's survival.

2. : A) always change the linking number in increments of 1. B) can act on single-stranded DNA circles. C) change the degree of supercoiling of a DNA molecule but not its linking number of DNA. D) occur in , but not in . E) require energy from ATP. Book Answer, but students pointed out that there is no correct answer

3. The SMC proteins (for structural maintenance of chromosomes) include cohesins and condensins, and are known to have all of the following properties except: A) A complete ATP . B) A hinge region C) activity to produce positive supercoils D) The ability to condense DNA E) Two coiled-coil domains

4 At replication forks in E. coli: A) DNA make endonucleolytic cuts in DNA. B) DNA primers are degraded by exonucleases. C) DNA topoisomerases make endonucleolytic cuts in DNA. D) RNA primers are removed by . E) RNA primers are synthesized by primase.

5. Which of these is not directly involved in methyl-directed mismatch repair in E. coli? A) DNA glycosylase B) DNA II C) DNA D) DNA III E) Exonuclease I

6. An alternative repair system by error-prone translesion DNA synthesis can result in a high mutation rate, because: A) alternative modified can be incorporated more readily. B) interference from the RecA and SSB proteins hinders the normal replication accuracy. C) replication proceeds much faster than normal, resulting in many more mistakes. D) the DNA involved cannot facilitate base-pairing as well as DNA polymerase III. E) the DNA polymerases involved lack exonuclease proofreading activities. Essay questions - 15 points each - Do any 5

7. Vocabulary What is the difference between.. An intron and an exon? An intron is an intervening piece of DNA that lies between Exons that are expressed.

A and a centromere? A centromere is a specialized piece of DNA at the center of the chromosome, while a are at either end of the chromosome. A linking number and a writhing number? A linking number gives the overall number of times one strand of DNA passes the plane define by the other strand and has an integer value. Oops, my bad. This edition of the text dropped writhing number, no points of for writhe. A writhing number tells about the overall crossing between two strands and can have non-integer values, roughly equivalent to supercoils. A plectonemic and a solenoidal supercoil? A plectonemic supercoil is a loose twist over a large distance. Solenoidal supercoils are tightly wrapped like a solenoid wire. A Histone and a nucleosome? A histone is a type of that is used to make nucleosomes.

Heterochromatin and Euchromatin? Heterochromatin is transcriptionally inactive DNAin a highly condensed structure. Euchromatin is transcriptionally active DNA that is a looser structure that is more available for transcription.

Condensin and cohesin? Condensins are proteins that maintain DNA in a condensed state as cells enter mitosis when separase is releasing the DNA from Cohesins. Cohesins are loaded onto the chromosomes during replication to keep sister chromatids together.

8.Explain why having negative supercoils can be useful to have in a piece of DNA. Can you think of any reasons why having positive supercoils would be useful? Negative supercoils make the DNA less twisted that it ‘wants’ to be. This can be useful in two ways. First, because the DNA is less twisted than it should be, it makes it easier for proteins to open up the DNA and get between the two strands. Second, supercoils, either positive or negative, are overall twists in the DNA structure that serve to make it more condensed and compact. There is no right or wrong answer for the positive supercoiling part of this question, but positive supercoiling would make the DNA harder to open up and also condense it so it would be useful when you are trying to condense DNA into a tight structure. This actually happens when Condensins bind to DNA.

-2- 9. When Eukariotic cells are not replicating, and recombinational repair is not feasible how are doubles strand breaks in the DNA repaired? Under these conditions the non-homologus end joining (NHEJ) mechanism is used. A diagram like figure 25-37 would help illustrate the mechanism. In this mechanism the proteins Ku70 and Ku80 first bind to the broken ends to serve as a scaffold for the other repair components. Next a kinase called DNA-PKcs and a nuclease called Artemis bind. The two broken ends now bind together and the repair begins. Phosphorylation of Artemis by DNA-PKcs makes activates it as an endouclease to remove 3' or 5' ends of the broken and a helicase startes to open up the two broken ends. This continues until a short piece of complementary DNA is found on the two broken strands that allows them to be annealed together. Any unpaired DNA is removed by Artemis and either DNA polymerse ì or ë is used to fill in the gaps in the DNA and a complex of XRCC4, XLF, and DNA ligase IV closes the in the DNA. Since the DNA is constrained the chromatin structure when this kind of repair occurs, the two pieces of DNA are usually very close together in sequence. While this mechanism can produce mutations, this type of repair is not used in cells that produce sperm or eggs, so the mutation is not passed on. In addition this type of repair is used in diploid cell, where presumably a second duplicate chromosome that has not beeqn damaged exists, so the effects of this kind of mutation are minimized.

10. Explain how DNA polymerase III works, and why is it so much more complicated that DNA polymerase I. The job of DNA pol III is to replicate both strand of the DNA simultaneously and to do it in a highly processive and accurate manner. While it is difficult to draw, a sketch like figure 25-9 of the DNA polymerse III would be a great place to start. While I don’t think I can expect you to know the names of all the individual subunits, identifying that the polymerase complex actually consists of a clamp loading complex attached to three different polymerase cores that bind Clamp proteins will probably help your explanation. A diagram like either figure 25-12 or 25-13 is almost necessary to explain how one of the three polymerase cores is holds on to one strand of the DNA and replicates it in the 5' 6 3' direction continuously. The other two cores switch off to replicate short fragments on the other strand so you can simultaneously replicate both strands at once. The other strand is replicated by putting a clamp on the strand, using the clamp loading complex, binding it to a primase and then to one of the polymerase cores and replicating that strand until you hit the double strand of DNA that was being replicated by the third polymerase core. The reason that DNA Pol I is simpler than DNA pol III is that DNA pol I only has to replicate a single strand of DNA in the 5'63' direction and it does not have to hang onto the DNA for 10000's of base pairs before it falls off, while DNA pol III has to replicate both strand of the DNA in the 5'63' direction even though one strand is oriended in the opposite direction and the polymerase had to stay bound to the DNA for 10000's of base pairs.

-3- 11. I have found a new organism. It is unique in that it synthesizes DNA from molecules that look like this:

Assuming the final DNA structure is the same, tell me about how assembling DNA from this monomer would be different than assembling it from the usual monomer. Would the reaction mechanism be the same? Would you still assemble the DNA in the 5'63' direction, etc.

Mostly what I was looking for is that since the phosphates here are attached to the 3' of the deoxyribose instead of the 5', DNA must be polymerized in the 3'65' direction instead of the normal 5'63' direction. This would reverse the direction of all polymerization and repair mechanisms we talked about in this chapter.

12. Tell me about the physical structure of a typical and how that structure is it encoded in the sequence of the antibody gene. In particular how can you create millions of difference from a limited number of antibody genes? A typical antibody is formed from 2 heavy chains and 2 light chains that form into a Y like structure. Both the heavy chains and the light chains have regions where the sequence is constant (unchanged) from one antibody to the next and regions that are highly variable. The variable regions are physically near the tip of the Y structure and are where the antibody binds to the antigen. The high variability in this antigen bindign region is what allows your body to fend off millions of different viruses and bacteria, and even come up with ways to bind to new variation of viruses and bacteria. The structure of the gene consists of about 300 tandem sequences for the variable region followed by 4 tandem sequences for an attachment region follows by the core or constant region. As a immuno cell matures it uses recombination to attach a single variable sequence to a single attachment sequence to a single constant region for both the heavy and light chains, so the cell ends up expressing a single uniqie antibody. If this cell finds and antigen to bind to, the cell will get expressed and used over and over to make more and more antibodies to that antigen. If the cell does not find an antigen, it simply dies off. Statistically the combination of 300 different variable regions with 4 different attachment regions will yield at least 1200 different light chains. In reality the recombination is is bit sloppy, and, as we learn in the next chapter there are additional sloppy splicing events to yield about 3000 different light chains. The heavy chains use a similar mechanism to give us about 5000 different heavy chains. 3000 x 5000 gives a total of about 15,000,000 different antibodies that can be expressed from these two genes.

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