8. Reproduction of chromatin requires assembly of nucleosomes

19.8 Reproduction of chromatin requires assembly of nucleosomes


The description of chromatin as a thread of duplex DNA coiled around a series of histone octamers to form nucleosomes is the crucial first step toward visualizing the state of the genetic material in the nucleus. This somewhat static view accounts for the structure of the individual subunit and (to some degree) for its relationship with the adjacent subunit. However, the organization of nucleosomes must be flexible enough to satisfy the various structural and functional demands made on chromatin.


Cyclical changes in packing affect the entire mass of euchromatin. During cell division, euchromatin must become more tightly packaged in mitotic chromosomes. The transition is likely to be controlled by changes in proteins that are widely distributed throughout chromatin.


Replication and transcription are local events that require some dispersion of structure. Replication occurs as a series of individual events in local regions (replicons), generating duplicate double-stranded DNA regions each associated with a set of histone octamers. The events involved in reproducing the nucleosome particle have yet to be defined. We should like to know what happens to the nucleosome during replication, and how new nucleosomes are assembled.


It seems inevitable that the separation of parental DNA strands must disrupt the structure of the 30 nm fiber. We should like to know the extent of this disruption. Is it confined to the immediate vicinity of the point where DNA is being synthesized, or does it extend farther? Are there discernible structural differences between regions that have replicated and those that have yet to do so?


The transience of the replication event is a major difficulty in analyzing the structure of a particular region while it is being replicated. The structure of the replication fork is distinctive. It is more resistant to micrococcal nuclease and is digested into bands that differ in size from nucleosomal DNA. This suggests that a large protein complex is engaged in replicating the DNA, but the nucleosomes reform more or less immediately behind as it moves along.




Figure 19.26 Replicated DNA is immediately incorporated into nucleosomes. Photograph kindly provided by S. MacKnight.

Reproduction of chromatin does not involve any protracted period during which the DNA is free of histones. Once DNA has been replicated, nucleosomes are quickly generated on both the duplicates. This point is illustrated by the electron micrograph of Figure 19.26, which shows a recently replicated stretch of DNA, already packaged into nucleosomes on both daughter duplex segments.


How histones associate with DNA to generate nucleosomes has been a vexed and confusing question. Do the histones preform a protein octamer around which the DNA is subsequently wrapped? Or does an H32 PH42 kernel bind DNA, after which H2A PH2B dimers are added?




Figure 19.27 In vitro, DNA can either interact directly with an intact (crosslinked) histone octamer or can assemble with the H32-H42 tetramer, after which two H2A-H2B dimers are added.

Self-assembly in vitro is a slow process, limited by the tendency of the assembling particles to precipitate. It is difficult to know which conditions mimic the physiological. Both pathways can be used in vitro to assemble nucleosomes, as illustrated in Figure 19.27.


Accessory proteins are involved in assisting histones to associate with DNA. Candidates for this role can be identified by using extracts that assemble histones and exogenous DNA into nucleosomes. Accessory proteins may act as "molecular chaperones" that bind to the histones in order to release either individual histones or complexes (H3 PH4 or H2A PH2B) to the DNA in a controlled manner. This could be necessary because the histones, as basic proteins, have a general high affinity for DNA. Such interactions allow histones to form nucleosomes without becoming trapped in other kinetic intermediates (that is, other complexes resulting from indiscreet binding of histones to DNA).


Attempts to produce nucleosomes in vitro began by considering a process of assembly between free DNA and histones. But nucleosomes form in vivo only when DNA is replicated. A system that mimics this requirement has been developed by using extracts of human cells that replicate SV40 DNA and assemble the products into chromatin. The assembly reaction occurs preferentially on replicating DNA. It requires an ancillary factor, CAF-1, that consists of >5 subunits, with a total mass of 238 kD. CAF-1 is recruited to the replication fork by PCNA, the processivity factor for DNA polymerase. This provides the link between replication and nucleosome assembly, ensuring that nucleosomes are assembled as soon as DNA has been replicated.


CAF-1 acts stoichiometrically, and functions by binding to newly synthesized H3 and H4 This suggests that new nucleosomes form by assembling first the H3 PH4 tetramer, and then adding the H2A PH2B dimers. The nucleosomes that are formed have a repeat length of 200 bp, although they do not have any H1 histone, which suggests that proper spacing can be accomplished without H1.




Figure 19.28 If histone octamers were conserved, old and new octamers would band at different densities when replication of heavy octamers occurs in light amino acids (part 1); but actually the octamers band diffusely between heavy and light densities, suggesting disassembly and reassembly (part 2).
Multiple figure

When chromatin is reproduced, a stretch of DNA already associated with nucleosomes is replicated, giving rise to two daughter duplexes. What happens to the pre-existing nucleosomes at this point? Are the histone octamers dissociated into free histones for reuse, or do they remain assembled? The integrity of the octamer can be tested by crosslinking the histones. Figure 19.28 summarizes the possible outcomes from an experiment in which cells are grown in the presence of heavy amino acids to identify the histones before replication. Then replication is allowed to occur in the presence of light amino acids. At this point the histone octamers are crosslinked and centrifuged on a density gradient. If the original octamers have been conserved, they will be found at a position of high density, and new octamers will occupy a low density position; but if the old histones have been released and then reassembled with newly synthesized histones, the octamers will have an intermediate density. Little material is found at the high density position, which suggests that histone octamers are not conserved.


The pattern of disassembly and reassembly is far from clear. It could be the case that the octamers are entirely dissociated into their constituent histones. However, one possibility is that the octamers lose one or both H2A PH2B dimers, which are replaced at random by new or old material. In this case, the H32 PH42 tetramer might be conserved. It is possible that a similar type of disruption occurs during transcription (see later). The H32 PH42 tetramer could have an ability to be transiently associated with a single strand of DNA during replication; it may in fact have an increased chance of remaining with the leading strand for reuse..




Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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