DNA REPLICATION
Learning Objectives for this Section
A. DNA Replication in Bacteria
In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing (def). Replication begins at a specific site in the DNA called the origin of replication.
DNA replication is bidirectional from the origin of replication (see Fig. 2). To begin DNA replication, unwinding enzymes called DNA helicases (def) cause the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks (def). These replication forks are the actual site of DNA copying (see Fig. 3). Helix destabilizing proteins bind to the single-stranded regions so the two strands do not rejoin. Enzymes called topoisimerases produce breaks in the DNA and then rejoin them in order to relieve the stress in the helical molecule during replication.
As the strands continue to unwind and separate in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand. As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. Actually, the nucleotides lining up by complementary base pairing are deoxynucleoside triphosphates, composed of a nitrogenous base, deoxyribose, and three phosphates. As the phosphodiester bond forms between the 5' phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand, two of the phosphates are removed providing energy for bonding (see Fig. 4). In the end, each parent strand serves as a template to synthesize a complementary copy of itself, resulting in the formation of two identical DNA molecules (see Fig. 5). During this process, the DNA molecules may attach to the cytoplasmic membrane and, as the cell elongates, the two DNA molecules are physically separated.
In reality, DNA replication is more complicated than this because of the nature of the DNA polmerases. DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl (OH) group of the 3' carbon of a nucleotide already in the chain. As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5' direction. Remember as mentioned above, each DNA strand has two ends. The 5' end of the DNA is the one with the terminal phosphate group on the 5' carbon of the deoxyribose; the 3' end is the one with a terminal hydroxyl (OH) group on the deoxyribose of the 3' carbon of the deoxyribose (see Fig. 6). The two strands are antiparallel, that is they run in opposite directions. Therefore, one parent strand - the one running 3' to 5' and called the leading strand (def) - can be copied directly down its entire length (see Fig. 7). However, the other parent strand - the one running 5' to 3' and called the lagging strand (def) - must be copied discontinuously in short fragments (Okazaki fragments) of around 100-1000 nucleotides each as the DNA unwinds.
In addition, DNA polymerase enzymes cannot begin a new DNA chain from scratch. They can only attach new nucleotides onto 3' OH group of a nucleotide in a preexisting strand. Therefore, to start the synthesis of the leading strand and each DNA fragment of the lagging strand, an RNA polymerase complex called a primosome or primase is required. The primase, which is capable of joining RNA nucleotides without requiring a preexisting strand of nucleic acid, first adds several comlementary RNA nucleotides opposite the DNA nucleotides on the parent strand. This forms what is called an RNA primer (see Fig. 8).
DNA polymerase III then replaces the primase and is able to add DNA nucleotides to the RNA primer (see Fig. 9). Later, DNA polymerase II digests away the RNA primer and replaces the RNA nucleotides of the primer with the proper DNA nucleotides to fill the gap (see Fig. 10). Finally, the DNA fragments themselves are hooked together by the enzyme DNA ligase (def) (see Fig. 7). Yet even with this complicated procedure, a 1000 micrometer-long macromolecule of tightly-packed, supercoiled DNA can make an exact copy of itself in only about 10 minutes time under optimum conditions, inserting nucleotides at a rate of about 1000 nucleotides per second!
There is a great deal of genetic information in the bacterial chromosome. For example Escherichia coli, the most studied of all bacteria, has a genome containing 4,639,221 base pairs, which code for at least 4288 proteins.
2. DNA Replication in Eukaryotes
As in prokaryotes, the linear chromosomes of eukaryotes replicate by strand separation and complementary base pairing (def) of free deoxyribonucleotides (def) with those on each parent DNA strand (see Fig. 4 and Fig. 5). As with prokaryotes, DNA replication in eukaryotic cells is bidirectional. However, unlike the circular DNA in prokaryotic cells that usually has a single origin of replication (see Fig. 2), the linear DNA of a eukaryotic cell contains multiple origins of replication (see Fig. 11).
As discussed earlier under prokaryotic DNA replication, DNA can only be synthesized in a 5' to 3' direction (see Fig. 5) and all DNA polymerase (def) requires a primer. To solve this problem, the ends of the linear eukaryotic DNA strands, called telomeres (def), have short, repetitive, noncoding DNA base sequences. A unique enzyme called telomerase binds to the telomeric DNA at the 3' end. The telomerase contains a small RNA template as a cofactor which is copied by DNA nucleotides to extend the 3' end. Once the extension is long enough, primase can assemble a short RNA primer on the lagging strand and DNA replication can proceed in a manner similar to the lagging strand of prokaryotic DNA. Once the chromosomes have replicated, the nucleus divides by mitosis. The eukaryotic cell cycle and mitosis will be discussed in the next tutorial.
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© Gary E. Kaiser
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Updated: September 7, 2004