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The authors refer to "epigenetically determined chromosomal states". In general, "epigenetic" refers to heritable/propagatable, often covalent modifications that affect the phenotype but not the genotype of an organism. Examples of this type of epigenesis include DNA methylation and histone acetylation, where methylation or acetylation changes in one cell generation are transmitted to the daughter generation. Another type of epigenesis is a propagatable conformational change in a protein. Prions are a good example of this - when a prion is mutated, it adopts a stable pathogenic conformation that can interact with normal prions and irreversibly convert them to the same pathogenic conformation. A third type of epigenesis (which happens to involve histone acetylation) is the inheritance of "chromosomal states", chromosomal structures that are either active or inactive. For example, certain regions of the genome are heterochromatic (transcriptionally inactive), and remain so in the next cell generation. After passage of the replication fork, chromatin must be assembled onto the newly replicated strands in such a manner as to preserve the pre-replication chromosomal state. This replication-coupled assembly, then, is a critical point for regulating inheritance of chromosomal structural information. CAF-1 appears to be involved in replication-coupled assembly both at the replication fork and at sites of DNA repair (e.g., nucleotide excision repair).

Nucleosome assembly is correlated with negative supercoils, because of the winding of the DNA around the core of histones.

You might want to refresh your memory on nucleosome structure by reviewing section 9.5 in your textbook (esp. pages 321-323). One technique used in this paper to determine the presence of nucleosomes is "micrococcal nuclease digestion" of CAF-1 treated DNA. This bacterial nuclease recognizes and incises the "linker" DNA regions between adjacent nucleosomes, releasing nucleosome units (mononucleosomes, di-, tri-, etc.) that can be visualized on agarose gels. The extent of digestion is dependent on the amount of added nuclease and incubation time.

To block or arrest DNA replication in the experiments shown in the paper, the authors use aphidicolin, a tetracyclic compound that inhibits eukaryotic DNA polymerases alpha, delta, and epsilon, but not beta or gamma.

An S100 extract is a cytosolic extract.

and now some questions...

1. Figure 1

a. (1 pt) Referring to Figure 1, the authors state that "the labeled, replicated DNA was supercoiled, but the unreplicated DNA was not". The authors correlate this with "preferential assembly" of nucleosomes on the replicated DNA strands. Explain in detail how this figure supports this statement, referring to both the autoradiogram and the EtBr-stained agarose gel.

b. (1 pt) What new information is demonstrated by the data in figure 1?

2. Figure 2

a. (.5 pt) Why did the authors allow the replication reaction to go for 40 minutes as opposed to a shorter time?

b. (.5 pt) The authors state that "the dependence of chromatin assembly on DNA replication could be due to a cis-acting mark left on the replicated DNA". What do you suppose they mean by "cis-acting mark"? What kinds of modifications can you think of that might constitute such a mark?

c. (0.5 pt) In testing for the possibility of cis-acting marks on DNA, explain in detail what the authors hoped to accomplish by including a spin column chromatography step and adding fresh S100 extract to the supercoiling reaction.

d. (.5 pt) The authors state that "the preferential supercoiling observed in Fig. 2 was due to formation of an array of nucleosomes...". Explain the assay they used to demonstrate this point. How and why do the data they obtained (as described in the text of their paper) support this idea?

3. Figure 3

a. (1 pt) Compare lanes 18-21 to lanes 25-28. What is different about these two sets of lanes, and what do these data suggest?

b. (.5 pt) The authors added a large excess of CAF-1 over RFC to exclude the "the possibility that RFC might be a competitive inhibitor of CAF-1 or that RFC might block CAF-1 activity by direct binding". Choose one of these possibilities and explain how addition of excess CAF-1 led them to exclude that possibility.

c. (1 pt) The authors state that "RFC...did not increase nicking of DNA (compare lanes 27 and 28 with 22, 23, or 24)." How do they know this? In other words, what would they have expected to see if RFC had nicking activity?

4. Figure 4

a. (.5 pt) Why is supercoiling not seen in Figure 4? (It's not because they ran the supercoiled bands off the gel!)

b. (1 pt) What data support the authors' contention that PCNA was being removed by RFC in this experiment?

5. (1 pt) Of the two experiments represented in Figures 5 and 6, which one demonstrated that PCNA is necessary for assembly of nucleosomes? Explain how.

6. (1 pt) Based on the data, the authors propose that PCNA serves as the DNA "imprint" that attracts CAF-1 to newly-replicated DNA strands, thereby facilitating replication-coupled chromatin assembly. Figure 8 illustrates the fact that there should be more PCNA on the lagging vs leading strands, implying that more nucleosomes could be loaded onto lagging vs leading strands. They speculate that asymmetric assembly of chromatin on the two sister chromatids might be involved in cell specialization during development. Clearly, though, such asymmetry would not always be desirable. What molecule discussed in this paper might regulate this effect? Explain.

7. (1 pt extra credit) Draw a double-stranded chromosome with three origins of replication spaced along it, both before and after replication. Indicate the 5' and 3' ends of each strand. Now draw in the distribution of PCNA that you might expect to see on each replicated chromosome, assuming this distribution has not been altered in the regulated fashion described above.

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