5–11 You are investigating DNA synthesis in tissue-culture cells, using 3H-thymidine to radioactively label the replication forks. By breaking open the cells in a way that allows some of the DNA strands to be stretched out, very long DNA strands can be isolated intact and examined. You overlay the DNA with a photographic emulsion, and expose it for 3 to 6 months, a procedure known as auto-radiography. Because the emulsion is sensitive to radioactive emissions, the 3H-labeled DNA shows up as tracks of silver grains. Because the stretching collapses replication
Figure Q5–2 Autoradiographic investigation of DNA replication in cultured cells (Problem 5–11). (A) Addition of 3H-labeled thymidine immediately after release from the synchronizing block. (B) Addition of 3H-labeled thymidine 30 minutes after release from the synchronizing block.
bubbles, the daughter duplexes lie side by side and cannot be distinguished from each other.
You pretreat the cells to synchronize them at the beginning of S phase. In the first experiment, you release the synchronizing block and add 3H-thymidine immediately. After 30 minutes, you wash the cells and change the medium so that the total concentration of thymidine is the same as it was, but only one-third of it is radioactive. After an additional 15 minutes, you prepare DNA for autoradiography. The results of this experiment are shown in Figure Q5–2A. In the second experiment, you release the synchronizing block and then wait 30 minutes before adding 3H-thymidine. After 30 minutes in the presence of 3H-thymidine, you once again change the medium to reduce the concentration of radioactive thymidine and incubate the cells for an additional 15 minutes. The results of the second experiment are shown in Figure Q5–2B.
A. Explain why, in both experiments, some regions of the tracks are dense with silver grains (dark), whereas others are less dense (light).
b.
In the first experiment, each track has a central dark section with light sections at each end. In the second experiment, the dark section of each track has a light section at only one end. Explain the reason for this difference.
C.
Estimate the rate of fork movement (μm/min) in these experiments. Do the estimates from the two experiments agree? Can you use this information to gauge how long it would take to replicate the entire genome?
5–12 If you compare the frequency of the sixteen possible dinucleotide sequences in the E. coli and human genomes, there are no striking differences except for one dinucleotide, 5ʹ-CG-3ʹ. The frequency of CG dinucleotides in the human genome is significantly lower than in E. coli and significantly lower than expected by chance. Why do you suppose that CG dinucleotides are underrepresented in the human genome?
5–13 With age, somatic cells are thought to accumulate genomic “scars” as a result of the inaccurate repair of double-strand breaks by nonhomologous end joining (NHEJ).
Estimates based on the frequency of breaks in primary human fibroblasts suggest that by age 70, each human somatic cell may carry some 2000 NHEJ-induced mutations due to inaccurate repair. If these mutations were distributed randomly around the genome, how many pro-tein-coding genes would you expect to be affected? Would you expect cell function to be compromised? Why or why not? (Assume that 2% of the genome—1.5% protein-coding and 0.5% regulatory—is crucial information.) 5–14 Draw the structure of the double Holliday junction that would result from strand invasion by both ends of the broken duplex into the intact homologous duplex shown in Figure Q5–3. Label the left end of each strand in the Holliday junction 5ʹor 3ʹ so that the relationship to the parental and recombinant duplexes is clear. Indicate how DNA synthesis would be used to fill in any single-strand gaps in your double Holliday junction.
(Problem 5–14).