I. MICROBIAL GENETICS
C. DNA REPLICATION
DNA is a long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides (def). A deoxyribonucleotide is composed of 3 parts: a molecule of the 5-carbon sugar deoxyribose, a nitrogenous base (def), and a phosphate group.
To synthesize the two chains of deoxyribonucleotides during DNA replication, the DNA polymerase enzymes involved 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. The covalent bond that joins the nucleotides is called a phosphodiester bond (def). Each DNA strand has what is called a 5' end and a 3' end. This means that one end of each DNA strand, called the 5' end (def) , will aways have a phosphate group attached to the 5' carbon of its terminal deoxyribonucleotide (see Fig. 1). The other end of that strand, called the 3' end (def) , will always have a hydroxyl (OH) on the 3' carbon of its terminal deoxyribonulceotide.
As will be seen in the next section, each parent strand, during DNA replication, acts as a template for the synthesis of the other strand by way of complementary base pairing. Complementary base pairing (def) refers to nucleotides with the base adenine hydrogen bonding only with nucleotides having the base thymine (A-T). Likewise, nucleotides with the base guanine can hydrogen bond only with nucleotides having the base cytosine (G-C). (As a result of this bonding, the DNA assumes its helical shape.) Therefore, the two strands of DNA are said to be complementary. Wherever one strand has an adenine-containing nucleotide, the opposite strand will always have a thymine nucleotide; wherever there is a guanine-containing nucleotide, the opposite strand will always have a cytosine nucleotide (see Fig. 1).
While the two strands of DNA are complementary, they are oriented in opposite directions to each other. One strand is said to run 5' to 3'; the opposite DNA strand runs antiparallel, or 3' to 5' (see Fig. 1).
We will now look at how DNA is replicated in both prokaryotic cells and eucaryotic cells.
1. 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. 1). 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. 6). 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. 7).
DNA polymerase III then replaces the primase and is able to add DNA nucleotides to the RNA primer (see Fig. 8). 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. 9). 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.
Some relationships between bacterial protein synthesis and bacterial enzymes and the control of bacteria with chemotherapeutic agents.
Some antimicrobials inhibit normal nucleic acid replication in bacteria.
1. The fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) work by inhibiting one or more of a group of enzymes called topoisomerase (def), enzymes needed for bacterial nucleic acid synthesis. For example, DNA gyrase (topoisomerase II), mentioned earlier in this unit, breaks and rejoins the strands of bacterial DNA to relieve the stress of the unwinding of DNA that occurs during DNA replication and transcription (def).
2. The sulfonamides and trimethoprim (co-trimoxazole - a combination of sulfamethoxazole and trimethoprim, sulfanilamide, etc.) block enzymes in the bacteria pathway required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine (see Fig. 17). This is done through a process called competitive antagonism whereby a drug chemically resembles a substrate in a metabolic pathway. Because of their similarity, either the drug or the substrate can bind to the substrate's enzyme. While the enzyme is bound to the drug, it is unable to bind to its natural substrate and that blocks that step in the metabolic pathway (see Fig. 18). Sulfonamides such as sulfamethoxazole tie up the first enzyme in the pathway, the conversion of para-aminobenzoic acid to dihydropteroic acid. Trimethoprim binds to the third enzyme in the pathway, an enzyme that is responsible for converting dihydrofolic acid to tetrahydrofolic acid. Without the tetrahydrofolic acid, the bacteria can not synthesize DNA or RNA.
3. Metronidazole is a drug that is activated by the microbial proteins flavodoxin and feredoxin found in
microaerophilc and anaerobic bacteria and certain protozoans. Once activated, the metronidazole puts
nicks in the microbial DNA strands.
2. DNA Replication in Eukaryotes and the Eukaryotic Cell Cycle
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 (def) (see Fig. 12 through 16).
The eukaryotic cell cycle is divided into two major phases: interphase and cell division.
A. Interphase
Ninety percent or more of the cell cycle is spent in interphase. During interphase, cellular organelles double in number, the DNA replicates, and protein synthesis occurs. The chromosomes are not visible and the DNA appears as uncoiled chromatin.
Interphase in a plant cell: see Fig. 17
Interphase in an animal cell: see Fig. 18
Interphase is divided into the following stages: G1, S, and G2.
1. G1 phase
During G1 phase, the period that immediately follows cell division, the cell grows and differentiates. New organelles are made but the chromosomes have not yet replicated in preparation for cell division.
2. S phase
DNA synthesis occurs during S phase. The chromosomes replicate in preparation for cell division.
3. G1 phase
During G2 phase, molecules that will be required for cell replication are synthesized.
B. Cell Division
Cell division consists of nuclear division and cytoplasmic division. Nuclear division is referred to as mitosis while cytoplasmic division is called cytokenesis.
1. Mitosis (nuclear division)
Mitosis is the nuclear division process in eukaryotic cells and ensures that each daughter cell receives the same number of chromosomes as the original parent cell. Mitosis can be divided into the following phases: prophase, metaphase, anaphase, and telophase.
a. Prophase
During prophase, the chromatin condenses and the chromosomes become visible. Also the nucleolus disappears, the nuclear membrane fragments, and the spindle appartus forms and attaches to the centromeres of the chromosomes.
Prophase in a plant cell: see Fig. 19 and Fig. 20
Prophase in an animal cell: see Fig. 21 and Fig. 22
b. Metaphase
During metaphase, the nuclear membrane fragmention is complete and the duplicated chromosomes line up along the cell's equator.
Metaphase in a plant cell: see Fig. 23
Metaphase in an animal cell: see Fig. 24
c. Anaphase
During anaphase, diploid sets of daughter chromosomes separate and are pushed and pulled toward opposite poles of the cell. This is accomplished by the polymerization and depolymerization of the microtubules that help to form the spindle apparatus.
Anaphase in a plant cell: see Fig. 25 and Fig. 26
Anaphase in an animal cell: see Fig. 27
d. Telophase
During telophase, the nuclear membrane and nucleoli reform, cytokinesis is nearly complete, and the chromosomes eventually uncoil to chromatin. Usually cytokinesis occurs during telophase.
Telophase in a plant cell: see Fig. 28 and Fig. 29
Telophase in an animal cell: see Fig. 30
2. Cytokinesis (cytoplasmic division)
During cytokinesis, the dividing cell separates into two diploid daughter cells. In animal cells, which lack a cell wall and are surrounded only by a cytoplasmic membrane, microfilaments of actin and myosin attached to the membrane form constricting rings around the central portion of the dividing cell and eventually divide the cytoplasm into two daughter cells. In the case of plant cells , which are surrounded by a cell wall in addition to the cytoplasmic membrane, carbohydrate-filled vesicles accumulate and fuse along the equator of the cell forming a cell plate that separates the cytoplasm into two daughter cells.
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