Eukaryotic DNA Replication

Mechanisms - The basic mechanisms of DNA replication are quite similar in eukaryotes and prokaryotes. DNA replication is semiconservative and is continuous on one strand and discontinuous on the other. As in prokaryotes, eukaryotic replication entails the assembly of short RNA primer molecules, elongation from the primers by a DNA polymerase, and (on the discontinuous strand) ligation of Okazaki fragments. A significant difference in eukaryotic and prokaryotic DNA replication is in the smaller size of the Okazaki fragments in eukaryotic cells - about 135 bases long, or about the size of the DNA on a nucleosome.

Enzymes - Eukaryotic cells contain five DNA polymerases. Three of them (polymerases , , and ) are used during S phase replication. Table 24.2 and Table 24.3 describe the properties of eukaryotic and prokaryotic DNA polymerases. As in prokaryotes, the replication complex also contains other proteins, including helicases and a number of accessory proteins called replication factors.

Assembly of Nucleosomes - Replication of the nuclear genome in eukaryotes presents some special problems. For example, the replication machinery must proceed through the complex nucleosomal structure of chromatin, which must apparently be dismantled and then reconstructed on the daughter DNA molecules as the replication machinery proceeds. Thus, not only must the DNA be faithfully copied, but also highly organized chromatin structure must be regenerated. It seems likely that nucleosomes from the parental chromatin strand are disassembled ahead of the replication fork and are then reassembled on one or the other of the daughter strands (Figure 28.17). Both preexisting and newly synthesized histones are used in the new nucleosomes. (H3/H4)2 tetramers and H2A/H2B dimers tend to remain intact, but other mixing is random.

Origins of Replication - Complications in dealing with the protein component in chromatin may explain why the rate of motion of a replication fork is about 10-fold slower in eukaryotes than in prokaryotes (compare with Table 24.1). The slow rate of fork motion (about 75 nucleotides/second) combined with the enormous size of eukaryotic genomes (roughly 108 base pairs), requires eukaryotic chromosomes to have many origins of replication--as many as several thousand on each chromosome. Replication proceeds bidirectionally from these origins, creating the replication "bubbles" shown in Figure 28.18. These bubbles grow independently until they finally merge and the whole chromosome has been copied. There is a tendency for transcriptionally active regions to begin replication early, whereas inactive regions replicate later.

In yeast, sequences called autonomously replicating sequences (ARSs) have been recognized as essential for the replication of plasmids. ARS sequences are typically several hundred base pairs in length with subsequences carrying copies of an 11 bp consensus sequence, called ACS (5' TTTTATATTTT 3'), which is absolutely required for function. ARSs have binding sites for the six-polypeptide origin binding factor as well as a strong affinity for the nuclear matrix.

Telomeres - Eukaryotic cells deal with the problem of completing the lagging strand of their linear chromosomes by the addition of telomeres at the ends of each chromosome. Telomeric DNA consists of simple tandemly repeated sequences like those shown in Table 28.2. Typically, one strand is G-rich, the other C-rich. These sequences are repeatedly added to the termini of chromosomal DNAs by enzymes called telomerases (Figure 28.19). This elongation allows room for a primer to bind and initiate synthesis on the other strand, maintaining the approximate length of the chromosome and preventing the loss of coding sequences. Telomerase must add nucleotides without the use of a DNA primer. This is probably accomplished through the existence, in each telomerase, of an essential RNA oligonucleotide that is complementary to the telomeric sequence being synthesized. Telomeres may have other important functions, too. For example, there is a strong correlation between aging, cell senescence, and low levels of telomerase. Conversely, cells in culture can be "immortalized" by introduction of active telomerase genes.

See also: Molecular Control of the Cell Cycle, The Cell Cycle, Eukaryotic DNA Polymerases (from Chapter 24)

INTERNET LINKS:

> 1. The Telomere Club

> 2. What are Telomeres?