Chapter 17 Nucleotides, Nucleic Acids, and Heredity

Genes, Exons, and Introns Gene: Gene: A segment of DNA that carries a base sequence that directs the synthesis of a particular protein, tRNA, or mRNA. There are many genes in one DNA molecule. In bacteria, the gene is continuous. In higher organisms, the gene is discontinuous. Exon: Exon: A section of DNA that, when transcribed, codes for a protein or RNA. Intron: Intron: A section of DNA that does not code for anything functional.

Genes, Exons, and Introns Figure The properties of mRNA molecules in prokaryotes cells during transcription and translation.

Genes, Exons, and Introns Figure The properties of mRNA in eukaryotic cells during transcription and translation

Replication of DNA The DNA in the chromosomes carries out two functions: replication (1) It reproduces itself. This process is called replication. (2) It supplies the information necessary to make all the RNA and proteins in the body, including enzymes. replication fork Replication begins at a point in the DNA called the origin of replication or a replication fork.

Replication of DNA Figure General features of the replication of DNA. The two strands of the DNA double helix are shown separating at the replication fork.

Replication of DNA The replication of DNA occurs in number of distinct steps. 1. Opening up of the superstructure of the chromosomes. One key step is this process is acetylation-deacetylation of lysine residues on histones. This reaction eliminates some of the positive charges on histones and weakens the strength of the DNA-histone interaction.

Replication of DNA Tropoisomerasesgyrases 2. Relaxation of Higher-Order Structures of DNA. Tropoisomerases (also called gyrases) temporarily introduce either single-or double strand breaks in DNA. Once the supercoiling is relaxed, the broken strands are joined together and the tropoisomerase diffuses from the location of the replication fork. helicases 3. Replication of DNA molecules starts with the unwinding of the double helix which can occur at either end or in the middle. Special unwinding proteins called helicases, attach themselves to one DNA strand and cause the separation of the double helix.

Replication of DNA 4. Primers/Primases Primers are short—4 to 15 nucleotides long—RNA oligonucloetides synthesized from ribonucleoside triphosphates. They are needed to initiate the primase- catalyzed synthesis of both daughter strands. 5. DNA Polymerase Once the two strands are separated at the replication fork, the DNA nucleotides must be lined up. In the absence of DNA polymerases, this alignment is extremely slow. The enzyme enables complementary base pairing with high specificity. While bases are being hydrogen bonded to their partners, polymerases join the nucleotide backbones.

Replication of DNA Okazaki fragments Along the lagging strand 3’—>5”, the enzymes can synthesize only short fragments, because the only way they can work is from 5’ to 3’. These resulting short fragments consist of about 200 nucleotides each, named Okazaki fragments after their discoverer. 6. Ligation The Okazaki fragments and any nicks remaining are eventually joined by DNA ligase.

How Do We Amplify DNA? To study DNA for basic and applied scientific purposes, we must have enough of it to work with. polymerase chain reaction (PCR) Millions of copies of selected DNA fragments can be made within a few hours with high precision by a technique called polymerase chain reaction (PCR). ◦ To use PCR, the sequence of a gene to be copied or at least a sequenced segment bordering the desired DNA must be known. ◦ In such a case, two primers that are complementary to the ends of the gene or to the bordering DNA can be synthesized. The primers are polynucleotides consisting of 12 to 16 nucleotides. When added to the target DNA segment, they hybridize with the end of each strand of the gene.

How Do We Amplify DNA? Figure Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence.

How Do We Amplify DNA? Figure Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence.

How Do We Amplify DNA A polymerase extends the primers in each direction as individual nucleotides are assembled and connected on the template DNA. In this way two copies are created. The two-step process is repeated (cycle 2) when the primers are hybridized with new strands and the primers extended again. At this point, four new copies have been created. The process is continued, and in 25 cycles, 2 25 or some 33 million copies can be made. This process is practical because of the discovery of heat-resistant polymerases isolated from bacteria that live in hot thermal vents on the sea floor. A temperature of 95°C is required to unwind the double helix to hybridize the primer to the target DNA.

How Is DNA repaired? The viability of cells depends on DNA repair enzymes that can detect, recognize, and remove mutations from DNA. Externally, UV radiation or highly reactive oxidizing agents, such as superoxide, may damage a base. Errors in copying or internal chemical reactions, such as deamination of a base,can create damage internally Deamination of cytosine turns it into uracil, which creates a mismatch. The former C-G base pair becomes a U-G mispair that must be removed. One of the most common base repair prepare means is called BER, base excision repair (Figure 17.15).

How Is DNA repaired? The BER pathway contains two parts: 1. A specific DNA glycolase (1) recognizes the damaged base and catalyzes the hydrolysis of the  -glycosidic bond between the uracil base base and the deoxyribose, then releases the damaged base completing the excision. The sugar-phosphate backbone is still intact. At the AP site (apurinic or apyrimidinic site) created in this way, (2) the backbone is cleaved by a second enzyme, endonuclease. A third enzyme, exonuclease (3), liberates the sugar-phosphate unit of the damaged site.

How Is DNA repaired? 2. In the synthesis step, the enzyme DNA polymerase (4) inserts the correct nucleotide, cytidine, and the enzyme DNA ligase seals (5) the backbone to complete the repair.

DNA Fingerprinting