Biol. 2320 - Keys to thought questions 3 & 4 Fall 2000

3a.       The point of this question was to think about why keeping the replication at the continuous and discontinuous strands together in space and time might be a good thing.  The most obvious answer was that the continuous strand half of the dimer would hold the other half in the correct position to find the sequence of primers used in the Okazaki fragments.  In other words, the fact that the continuous strand part of the polymerase holds on and keeps moving into the replication fork makes it possible for the discontinuous strand part to let go and then grab o again in the right place.

            Another good answer was that coordination of the synthesis of the two strands would facilitate the orderly termination of a round of replication.  If the two strands were replicating independently, and either one got a head of the other, there would have to be a mechanisms to stop the one that got ahead or it could overshoot and start re-replicating a stretch that had already been done.

            Simply re-stating how the system works to facilitate the formation of Okazaki fragments did not address the question.  Stating that having the strands replicate separately would some how be more likely to lead to damage or mutation was worth only partial credit unless you provided a specific example.

3b.       There were 3 correct answers: 1) The heavy/light N DNA incorporated into the Okazaki fragments was exactly the same as the continuous strand and so would be the same density, and seen as such by M & S.  2) M & S separated double stranded DNA by centrifugation so the Okazaki fragments would have moved with the parent DNA and thus not been seen.  3) Okazaki used a different density gradient centrifugation method that resolved molecules by size as opposed to just density (this information was in legends of figures 13.3 and 13.10, where it was mentioned that M & S used CsCl and Okazaki used sucrose as the gradient media respectively).

            Stating that M & S used cells in which replication was completed, or in which the Okazaki fragments were already ligated involved making an inaccurate assumption.  In bacterial cells replication occurs continuously.  Remember the theta structure Dr. Rushing showed where the origin of replication had “fired” again before the previous round of replication was complete.  Hence there would always be Okazaki fragments present.

Invoking the difference in labeling of the DNA (that M & S used just density labeling and Okazaki used radioactive labels) missed the point that Okazaki still separated the DNA by density.

4a.       If you chose BER as more similar you would want to argue that BER works solely on the base that is lesion, and that the glycosylase is like the photolyase in that it acts directly on the base that is the problem.

            If you chose NER you would want to argue that the detection system worked by identifying a large distortion in the DNA structure (specifically in this case, the TT dimer).

4b.       If you chose NER as dissimilar you would want to state that there is no helicase activity, no nuclease activity and no polymerase activity involved in photoreactivation and all of these are used in NER.

            If you chose BER as dissimilar you would want to state that there is no polymerase activity, that no base is removed, there is no nucelase, no phosphodiesterase, and no ligase activity.

4c.       The 3 enzymes in common were: DNA endonuclease; DNA polymerase and DNA ligase.  The endonuclease cuts the DNA once in BER and twice in NER, in both cases to remove DNA to be replaced by a polymerase.  The polymerase replaces the removed region.  The ligase rejoins the gap between new and original DNA.

            The helicase is specific to NER.  It unwinds the DNA between the two nicks caused by the endonuclease, causing the release of the damaged/mispaired base.  The glycosylase and phosphodiesterase are specific to BER.  The glycosylase removes the chemically modified base.  The phosphodiesterase removes the sugar-phosphate component of the nucleotide that has had its base removed.

4d.       Transcribed regions of DNA might be more susceptible to damage because they are constantly being “worked on” by the transcription machinery.  DNA binding proteins interact with the bases, distorting normal base-pairing and bending the DNA.  RNA polymerase opens up regions of single stranded DNA, which may be unstable.  DNA is forced to base-pair with RNA which may lead to chemical changes in the bases.