Compared with the situation in budding yeast, the determinants of replication origins in other eukaryotes have been difficult to discover. It has been possible to identify specific human DNA sequences, each several thousand nucleotide pairs in length, that are sufficient to serve as replication origins. These origins continue to function when moved to a different chromosomal region by recombinant DNA methods, as long as they are placed in a region where the chromatin is relatively uncondensed. However, comparisons of such DNA sequences have not revealed specific DNA sequences that mark origins of replication.
Despite this, a human ORC that is very similar to the yeast ORC binds to origins of replication and initiates DNA replication in humans. Many of the other proteins that function in the initiation process in yeast likewise have central roles in humans. It therefore seems likely that the yeast and human initiation mechanisms are similar in outline, but chromatin structure, transcriptional activity, or some property of the genome other than a specific DNA sequence has the central role in attracting ORC and specifying mammalian origins of replication. These ideas could also help to explain how a given mammalian cell chooses which of the many possible origins to use when it replicates its genome and how this choice could differ from cell to cell. Clearly, we have a great deal to discover about the fundamental process of DNA replication initiation.
New Nucleosomes Are Assembled Behind the Replication Fork
Several additional aspects of DNA replication are specific to eukaryotes. As discussed in Chapter 4, eukaryotic chromosomes are composed of roughly equal mixtures of DNA and protein. Chromosome duplication therefore requires not only the replication of DNA, but also the synthesis and assembly of new chromosomal proteins onto the DNA behind each replication fork. Although we are far from understanding this process in detail, we are beginning to learn how the fundamental unit of chromatin packaging, the nucleosome, is duplicated. The cell requires a large amount of new histone protein, approximately equal in mass to the newly synthesized DNA, to make the new nucleosomes in each cell cycle. For this reason, most eukaryotic organisms possess multiple copies of the gene for each histone. Vertebrate cells, for example, have about 20 repeated gene sets, most containing the genes that encode all five histones (H1, H2A, H2B, H3, and H4).
Unlike most proteins, which are made continuously, histones are synthesized mainly in S phase, when the level of histone mRNA increases about fiftyfold as a result of both increased transcription and decreased mRNA degradation. The major histone mRNAs are degraded within minutes when DNA synthesis stops at the end of S phase. The mechanism depends on special properties of the 3ʹends of these mRNAs, as discussed in Chapter 7. In contrast, the histone proteins themselves are remarkably stable and may survive for the entire life of a cell. The tight linkage between DNA synthesis and histone synthesis appears to reflect a feedback mechanism that monitors the level of free histone to ensure that the amount of histone made exactly matches the amount of new DNA synthesized.
As a replication fork advances, it must pass through the parental nucleosomes. In the cell, efficient replication requires chromatin remodeling complexes (discussed in Chapter 4) to destabilize the DNA–histone interfaces. Aided by such complexes, replication forks can transit even highly condensed chromatin efficiently.