Eukaryotic DNA Replication

EUKARYOTIC DNA REPLICATION

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been noted. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H and flap endonuclease-1 (FEN1) rather than by a DNA polymerase (Figure 29.21).

Figure 29.21 Proteins and their function in eukaryotic replication. ORC = origin recognition complex, MCM = minichromosome maintenance (complex), RPA = replication protein A, PCNA = proliferating cell nuclear antigen.

A. Eukaryotic cell cycle

The events surrounding eukaryotic DNA replication and cell division (mitosis) are coordinated to produce the cell cycle (Figure 29.22). 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 period (G₂ phase, or Gap₂) 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 (Cdks).]

Figure 29.22 The eukaryotic cell cycle. [Note: Cells can leave the cell cycle and enter a reversible quiescent state called G0.]

B. Eukaryotic DNA polymerases

At least five high-fidelity eukaryotic DNA polymerases (pol) 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 (Figure 29.23).

Figure 29.23 Activities of eukaryotic DNA polymerase (pol) *3 →5 exonuclease activity.

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 I →3I 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 d: Pol ε is recruited to complete DNA synthesis on the leading strand, whereas pol d elongates the Okazaki fragments of the lagging strand, each using 3I →5I 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 subunit of DNA pol III does in E. coli, thus ensuring high processivity.]

3. Pol β and pol γ: Pol β is involved in “gap filling” in DNA repair (see below). Pol γ replicates mitochondrial DNA.

C. Telomeres

Telomeres are complexes of noncoding 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 of Cs and As. The GT-rich strand is longer than its CA complement, leaving ssDNA a few hundred nucleotides in length at the 3I -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 5I -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 other stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the presence of a ribonucleoprotein, telomerase, which maintains telomeric length in these cells.

2. Telomerase: This complex contains a protein (Tert) that acts as a reverse transcriptase and a short piece of RNA (Terc) that acts as a template. The CA-rich RNA template base-pairs with the GT-rich, single-stranded 3I -end of telomeric DNA (Figure 29.24). The reverse transcriptase uses the RNA template to synthesize DNA in the usual 5 I →3I direction, extending the already longer 3I -end. Telomerase then translocates to the newly synthesized end, and the process is repeated. Once the GT-rich strand has been lengthened, primase activity of DNA pol α can use it as a template to synthesize an RNA primer. The RNA primer is extended by DNA pol α, and then removed.

Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres provides insight into the biology of aging and cancer.

Figure 29.24 Mechanism of action of telomerase. T= thymine; A = adenine; C = cytosine; G = guanine; pol = polymerase.

D. Reverse transcriptases

As seen with telomerase, reverse transcriptases are RNA-directed DNA polymerases. A reverse transcriptase is involved in the replication of retroviruses, such as human immunodeficiency virus (HIV). These viruses carry their genome in the form of ssRNA molecules. Following infection of a host cell, the viral enzyme reverse transcriptase uses the viral RNA as a template for the 5I →3I synthesis of viral DNA, which then becomes integrated into host chromosomes. Reverse transcriptase activity is also seen with transposons, DNA elements that can move about the genome. In eukaryotes, such elements are transcribed to RNA, the RNA is used as a template for DNA synthesis by a reverse transcriptase encoded by the transposon, and the DNA is randomly inserted into the genome. [Note: Transposons that involve an RNA intermediate are called retrotransposons or retroposons.]

E. Inhibition of DNA synthesis by nucleoside analogs

DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been modified on the sugar portion (Figure 29.25). For example, removal of the hydroxyl group from the 3I -carbon of the deoxyribose ring as in 2I ,3I - dideoxyinosine ([ddI] also known as didanosine), or conversion of the deoxyribose to another sugar, such as arabinose, prevents further chain elongation. By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses. Cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine arabinoside (vidarabine, or araA) is an antiviral agent. Substitution on the sugar moiety, as seen in zidovudine (AZT, ZDV), also terminates DNA chain elongation. [Note: These drugs are generally supplied as nucleosides, which are then converted to the active nucleotides by cellular kinases.

Figure 29.25 Examples of nucleoside analogs that lack a 3I -hydroxyl group. [Note: ddI is converted to its active form (ddATP).]