Question

In an article entitled "Nucleosome Positioning at the Replication Fork," Lucchini and others (2002. EMBOJ. 20: 7294-7302) state, "both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands." Given this statement, how would one compare the distribution of nucleosomes and DNA in newly replicated chromatin? How could one experimentally test the distribution of nucleosomes on newly replicated chromosomes?

Short answer

Answer: In newly replicated chromatin, the distribution of nucleosomes can be similar to the pre-replicated chromatin because of the rapid positioning of nucleosomes. This would lead to proper compaction, similar accessibility to regulatory regions, and maintenance of epigenetic modifications. To test this distribution, experimental techniques like Chromatin Immunoprecipitation (ChIP), Micrococcal Nuclease (MNase) digestion followed by DNA sequencing, and Click Chemistry and Nucleosome Tracing can be employed.

Step-by-step solution

1. Introduction to Nucleosomes and Chromatin

Nucleosomes are the basic units of chromatin, which are complexes of DNA, histone proteins, and other non-histone proteins. They consist of a histone octamer (two copies each of histones H2A, H2B, H3, and H4) around which the DNA wraps approximately 1.7 times. Nucleosomes function to organize and compact the DNA within the cell nucleus and play an important role in the regulation of gene expression.

2. Comparing the Distribution of Nucleosomes and DNA in Newly Replicated Chromatin

As stated in the article, both old and new nucleosomes rapidly position themselves on the newly replicated DNA strands. The distribution of nucleosomes on newly replicated DNA could follow similar patterns as before replication, due to the rapid positioning of nucleosomes. This would result in proper compaction of the chromatin, similar accessibility to regulatory regions, and the maintenance of epigenetic modifications from the parent strand to the daughter strands.

3. Experimental Techniques to Test Nucleosome Distribution

There are several experimental techniques that could be used to test the distribution of nucleosomes on newly replicated chromosomes: a) Chromatin Immunoprecipitation (ChIP): ChIP is a technique that involves cross-linking DNA and associated proteins (nucleosomes), shearing the DNA into smaller fragments, and immunoprecipitating the protein-DNA complexes using antibodies specific to histone proteins. The precipitated DNA can then be sequenced to identify the locations of nucleosomes. By performing ChIP on newly replicated chromatin, we can identify the distribution of nucleosomes on the new DNA. b) Micrococcal Nuclease (MNase) Digestion followed by DNA Sequencing: MNase is an enzyme that preferentially cleaves the DNA between nucleosomes. By digesting chromatin from newly replicated cells and then sequencing the resulting DNA, we can identify the locations of nucleosomes. c) Click Chemistry and Nucleosome Tracing: Click chemistry can be used to incorporate modified histones into newly synthesized nucleosomes. The modified histones can be tracked using microscopy or other detection techniques to determine the distribution of nucleosomes in newly replicated chromatin.