The events surrounding eukaryotic DNA replication and cell division (mitosis) are coordinated to produce the cell cycle (Fig. 30.22). The period preceding replication is called the G1 phase (Gap 1). DNA replication occurs during the S (synthesis) phase. Following DNA synthesis, there is another phase (G2, or Gap 2) before mitosis (M). Cells that have stopped dividing, such as mature T lymphocytes, are said to have gone out of the cell cycle into the G0 phase. Such quiescent cells can be stimulated to reenter the G1 phase to resume division. [Note: The cell cycle is controlled at a series of checkpoints that prevent entry into the next phase of the cycle until the preceding phase has been completed. Two key classes of proteins that control the progress of a cell through the cell cycle are the cyclins and cyclin-dependent kinases (Cdk).]
B. Eukaryotic DNA polymerases
At least five high-fidelity eukaryotic DNA pols have been identified and categorized on the basis of molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act. They are designated by Greek letters rather than by Roman numerals (Fig. 30.23).
1.
Pol α: Pol α is a multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. The primase subunit synthesizes a short RNA primer that is extended by the 5′→3′ polymerase activity of pol α, generating a short piece of DNA. [Note: Pol α is also referred to as pol α/primase.] 2.
Pol ε and pol δ: Pol ε is recruited to complete DNA synthesis on the leading strand, whereas pol δ elongates the Okazaki fragments of the lagging strand, each using 3′→5′ exonuclease activity to proofread the newly synthesized DNA. [Note: DNA pol ε associates with proliferating cell nuclear antigen (PCNA), a protein that serves as a sliding DNA clamp in much the same way the β subunits of DNA pol III do in E. coli, thus insuring high processivity.] 3.
Pol β and pol γ: Pol β is involved in gap filling in DNA repair. Pol γ replicates mitochondrial DNA.
C. Telomeres
Telomeres are complexes of DNA plus proteins (collectively known as shelterin) located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases, and allow repair systems to distinguish a true end from a break in dsDNA. In humans, telomeric DNA consists of several thousand tandem repeats of a noncoding hexameric sequence, AGGGTT, base-paired to a complementary region containing C and A. The G-rich strand is longer than its C-rich complement, leaving ssDNA a few hundred nucleotides in length at the 3′end. The single-stranded region is thought to fold back on itself, forming a loop structure that is stabilized by protein.
1.
Telomere shortening: Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5′-end of the lagging strand, there is no way to fill in the remaining gap with DNA. Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the ribonucleoprotein telomerase, which maintains telomeric length in these cells.
2.