Mammalian Chromosomal Organization

The human genome contains approximately 109 base pairs of DNA. The total DNA is contained in 46 double-stranded

DNA pieces complexed with proteins to form chromosomes. The diploid human genome, therefore, is contained in 46 chromosomes: two of each of the 22 autosomal chromosomes, plus either two X chromosomes, in females, or one X and one Y chromosome, in males. Since the length of each helical turn of a double-stranded DNA molecule is 3.4 nm, consisting of ten bases, the length of the total genomic DNA in each cell measures approximately 1m in length. For each cell to contain this long molecule, the double-stranded DNA must be compressed. A complex of eight basic histones (two copies of histone 2 [H2], H3, H4, and H5) package the DNA.9 The histone complex contains positively charged amino acids that bind to 146 bases of negatively charged DNA. Histones fold the DNA either partially or tightly, resulting in compression of the DNA strand. Tight folding of the DNA condenses the DNA into heterochromatin. Following packaging and condensation, the nucleic acid strand widens from 2nm to 1400 nm, with extensive overall shortening of the nucleic acid in the metaphase chromosome. Light microscopy easily permits the visualization of condensed metaphase chromosomes.

Less-condensed DNA binds histone 1 (H1) proteins or other sequence-specific DNA-binding molecules. Some of these DNA-binding molecules regulate gene expression (discussed later in this chapter). In contrast, tightly condensed chromosomes lack the "open spaces" for binding of regulatory proteins and prevent gene expression from tightly condensed DNA regions. These proteins may also prevent access to nucleic acid probes or primers for molecular diagnostic tests. As a result, many DNA extraction protocols include a protein-digestion step to liberate the DNA from these DNA-binding proteins. Removal of these proteins facilitates hybridization with short pieces of nucleic acid, such as primers or probes.

DNA Replication

Eukaryotic DNA Replication

The replication of DNA is a complex process requiring specific physiological temperatures and a host of proteins. As mentioned previously, molecular diagnostic techniques rely on the ability to denature or melt a double-stranded DNA template. Using chemical or physical conditions, separation of DNA strands can be accomplished with alkali or high temperatures (i.e., 95°C). Under physiological conditions, dissociation of DNA strands for replication is accomplished by numerous enzymes, such as helicases and topoisomerases. The region of transition from the double-stranded to separated single-stranded DNA is called the replication fork. The replication fork moves along the double-stranded DNA molecule as replication proceeds. At the replication fork, various primases, initiating proteins, and polymerases bind to the original or parental DNA strands and generate new daughter strands. Known collectively as a replisome, these enzymatic activities generate

Figure 1-3. DNA replication. Replication fork depicting the leading and lagging strands and the numerous proteins and Okazaki fragments involved with replication. (Reprinted from Leonard D. Diagnostic Molecular Pathology, copyright 2003, with permission from Elsevier.)

two new nucleic acid strands that are complementary to and base paired with each of the original two template or parent DNA strands. This replication process is known as semiconservative because each resulting double-stranded DNA molecule consists of one new and one old DNA strand (Figure 1-3).

Polymerases function to synthesize new nucleic acid molecules from nucleotide building blocks. The sequence of the new strand is based on the sequence of an existing nucleic acid molecule, and the polymerase adds nucleotides according to the order of the bases of the parent strand, using G : C and A : T pairing. The new strand is antiparallel to the parent strand and is synthesized in a 5' to 3' direction. Of the two parent strands of genomic DNA, one strand (called the leading strand) can be read by the polymerase continuously in a 3' to 5' direction, with the new strand generated in a continuous 5' to 3' direction. In contrast, the opposite strand (known as the lagging strand) cannot be read continuously by the polymerase. The replication fork moves along the lagging strand in a 5' to 3' direction, and polymerases synthesize only by reading the parent strand in a 3' to 5' direction while synthesizing the new strand in a 5' to 3' direction. Therefore, synthesis cannot proceed continuously along the lagging strand, which must be copied in short stretches primed from RNA primers and forming short DNA fragments known as Okazaki fragments. The new strand complementary to the lagging strand is formed by removal of the RNA primer regions and ligation of the short DNA fragments into a continuous daughter strand complementary to the lagging strand. Discontinuous 3' to 5' replication results in the progressive loss of ends of the chromosomes known as telomeres in normal cells. Some malignant cells retain telomerase activity that permits the addition of these terminal telomeric sequences to the chromosomes.

While replication requires many proteins, the poly-merase determines the speed and accuracy of new strand synthesis. The rate that the four nucleotides are polymerized into a nucleic acid chain defines the processivity of the enzyme. The processivity of most polymerases approximates 1000 bases per minute.

Table 1-4. Fidelity of Various Polymerases

Polymerase

Error Rate

pol ß*

8 x 10-4

pol a*

1 x 10-4

pol e*

1.7-4 x 10-5

Pfut

1.3 x 10-6

Deep ventt

2.7 x 10-6

Ventt

2.8 x 10-6

Taqt

8 x 10-6

UITmat

55 x 10-6

Klenowi

1-10 x 10-7

HIV reverse transcriptase

6-30 x 10-4

*Reference 37.

tReference 38.

i Reference 39.

The fidelity of the polymerase refers to the accuracy of the enzyme to incorporate the correct complementary bases in the newly synthesized DNA. Incorporation of incorrect bases or other replication errors can result in cell death or oncogenesis. The error rate of polymerases varies widely from 1 in 1200 to 1 in 1,000,000 bases (Table 1-4). To correct the erroneous incorporation of bases or other replication errors, protein complexes proofread and correct synthesis errors. In normal cells, the cell cycle pauses to facilitate error repair in the G2 phase of the cell cycle (Figure 1-4). Malignant cells may not pause to allow for error correction, resulting in the accumulation of damaged or mutated DNA.

The complexity of the biochemical reactions necessary for replicating eukaryotic nuclear DNA demonstrates a high degree of regulation for generating two strands from one replication fork. In addition to these complexities, replication in eukaryotic cells occurs at multiple origins of

Figure 1-4. Cell cycle. The clear panels are the ordered phases of mitosis (M phase), while the gray and black panels are the ordered stages of interphase. P, prophase; PM, prometaphase; MET, metaphase; A,anaphase; T,telophase; G1,gap 1; S, DNA synthesis; G2, gap 2.

replication (Ori). These multiple sites grow progressively until the newly generated strands join to form complete chromosomal-length DNA.

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