| DNA is compacted into chromosomes
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| In eukaryotes, DNA is arranged in linear segments termed chromosomes. Each chromosome contains between 48 million and 240 million base pairs. The B-form of DNA has a contour length of 3.4 Å per base pair. Therefore, chromosomes have contour lengths of 1.6-8.2 cm, which is much larger than a cell. To fit within the nucleus, DNA is precisely condensed (>8000-fold).
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In the native chromosome, DNA is complexed with RNA and an approximately equal mass of protein. These DNA-RNA-protein complexes are termed chromatin. The majority of the proteins in chromatin are histones. Histones are a highly conserved family of proteins that are involved in the packing and folding of DNA within the nucleus. There are five classes of histones, termed H1, H2A, H2B, H3, and H4. They are all rich (>20%) in positively charged, basic amino acids (lysine and arginine). These positive charges interact with the negatively charged, acidic phosphate groups of the DNA strands to reduce electrostatic repulsion and permit tighter DNA packing.
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| BIOCHEMISTRY OF DNA POLYMERASES |
| There are several different DNA polymerases in the cell. DNA polymerase III, the principal DNA-replication enzyme, has the highest rate of DNA polymerization. DNA polymerase I is involved in excision repair and in the removal of RNA primers from Okazaki fragments. |
| DNA polymerase III is a complex protein, with at least 10 distinct subunits, ranging in size from 12 kDa to 130 kDa. The action of DNA polymerase III requires a single-stranded template and a primer, either DNA or RNA. After binding to the template-primer complex, the enzyme first determines which nucleotide is complementary to the next available template nucleotide and allows that nucleotide to bind to the active site. The elongation reaction occurs when the 3'-hydroxyl of the previous base attacks the 5'-phosphate of the incoming deoxynucleotide triphosphate (dNTP). Pyrophosphate is released. This process results in an elongation of the DNA strand by one nucleotide. Then, in a process termed proof-reading, the enzyme checks whether the newly incorporated base can form an allowed Watson-Crick base pair (i.e. AT, TA, CG, or GC) with the opposing base on the template strand. If the base pair is not allowed, the enzyme removes the last added nucleotide and repeats the process. If the base pair is allowed, the enzyme advances one nucleotide along the template strand and repeats the process. The ability of DNA polymerase III to proofread DNA sequences and replace mismatched nucleotides serves to maintain the high fidelity of DNA replication. |
| The histone proteins associate into a complex termed a nucleosome (Fig. 30.4). Each of these complexes contain two
molecules each of H2A, H2B, H3, and H4 and one molecule of H1. The nucleosome protein complex is encircled with about 200 base pairs of DNA that form two coils around the nucleosome core. The H1 protein associates with the outside of the nucleosome core to stabilize the complex. By forming nucleosomes, the packing density of DNA is increased by a factor of about seven-fold.
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| The nucleosome particles themselves are also organized into other, more tightly packed structures, termed 300 Å chromatin filaments. These filaments are constructed by winding the nucleosome particles into a spring-shaped solenoid structure with about six nucleosomes per turn (Fig. 30.4). The solenoid is stabilized by head-to-tail associations of the H1 histones.
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| Finally, the chromatin filaments are compacted into the mature chromosome that is associated with a nuclear scaffold. The nuclear scaffold is about 0.4 mm in diameter and forms the core of the chromosome. The filaments are dispersed around the scaffold to form radial loops about 0.3 mm in length. The final diameter of a chromosome is about 1 mm.
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| Figure 30.4 Structures involved in chromosome packaging. (A) The nucleosome core is composed of two subunits each of H2A, H2B, H3, and H4. The core is twice wrapped with DNA, and the H1 histone binds to the completed complex. (B) The 300 Å chromatin filament is formed by wrapping the nucleosomes into a spring-shaped solenoid. (C) The chromosome is composed of the 300 Å filaments, which bind to a nuclear scaffold, forming large loops of chromatin material. (D) The end view of a chromosome shows the central nuclear scaffold surrounded by the radial loops of chromatin. The diameter of a chromosome is about 1 μm. |
| The ends of the chromosomes are composed of unique DNA sequences called telomeres. These structures consist of tandem repeats of short, G-rich, species-specific oligonucleotides. In humans, the repeated sequence is TTAGGG. Telomeres can contain as many as 1000 copies of this sequence. During the synthesis of telomeres, the enzyme telomerase adds the preformed hexanucleotide repeats onto the 3'-end of the chromosome. There is no requirement for a DNA template in the elongation of telomeres at chromosome ends.
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