Experimental procedure for studying how nucleosomes dissociate and reassociate during replication

Techniques of fluorescence microscopy, which are capable of revealing active sites of DNA synthesis, show that most replication in the nucleus of a eukaryotic cell takes place at a limited number of fixed sites, often referred to as replication factories. Time-lapse micrographs reveal that newly duplicated DNA is extruded from these particular sites. Similar results have also been obtained with bacterial cells.

DNA synthesis at the ends of chromosomes A fundamental difference between eukaryotic and bacterial replication arises because eukaryotic chromosomes are linear and thus have ends. As already stated, the 3'-OH group needed for replication by DNA polymerases is provided at the initiation of replication by RNA primers that are synthesized by primase. This solution is temporary, because eventually the primers must be removed and replaced by DNA nucleotides. In a circular DNA molecule, elongation around the circle eventually provides a 3'-OH group immediately in front of the primer (IFigure 12.19a). After the primer has been removed, the replacement DNA nucleotides can be added to this 3'-OH group.

In linear chromosomes with multiple origins, the elongation of DNA in adjacent replicons also provides a 3'-OH group preceding each primer (I Figure 12.19b). At the very end of a linear chromosome, however, there is no adjacent stretch of replicated DNA to provide this crucial 3'-OH group. Once the primer at the end of the chromosome has been removed, it cannot be replaced with DNA nucleotides, which produces a gap at the end of the chromosome (iFigure 12.19c), suggesting that the chromosome should become progressively shorter with each round of replication. The chromosome would be shortened each generation, leading to the eventual elimination of the entire telomere, desta-bilization of the chromosome, and cell death. But chromosomes don't become shorter each generation and destabilize; so how are the ends of linear chromosomes replicated?

The ends of chromosomes—the telomeres—possess several unique features, one of which is the presence of many copies of a short repeated sequence. In the protozoan Tetrahymena, this telomeric repeat is CCCCAA (see Table 11.2), with the G-rich strand typically protruding beyond the C-rich strand ( Figure 12.20a):

end of 5'-CCCCAA toward

chromosome 3 '-GGGGTTGGGGTT centromere

(a) Circular DNA

Primer oh J^Or

Template-^ DNA

Replication around the circle provides a 3'-OH group in front of primer, onto which nucleotides can be added when the primer is replaced.

Replication around circle

(a) Circular DNA

Primer oh J^Or

Template-^ DNA

Replication around the circle provides a 3'-OH group in front of primer, onto which nucleotides can be added when the primer is replaced.

Replication around circle

^Primers at the ends of chromosomes cannot be replaced, because there is no adjacent 3'-OH to which DNA nucleotides can be attached.

(c) End of a linear chromosome

Synthesis of primer

Elongation of DNA

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