Lesson 6 Medicine 2024-2025 PDF

Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, genetic information is propagated into the next generation exclusively by germ-line cells (red). This cell lineage includes the specialized reproductive cells—the gametes (eggs and sper...

Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, genetic information is propagated into the next generation exclusively by germ-line cells (red). This cell lineage includes the specialized reproductive cells—the gametes (eggs and sperm, half circles)—which contain only half the number of chromosomes as that contained in the other cells in the body (full circles). When two gametes come together during fertilization, they form a fertilized egg, or zygote (purple), which once again contains a full set of chromosomes. The zygote gives rise to both germ-line cells and somatic cells (blue). Somatic cells form the body of the organism but do not contribute their DNA to the next generation. DNA acts as a template for its own replication. Because the nucleotide A will successfully pair only with T, and G with C, each strand of a DNA double helix—labeled here as the S strand and its complement, the S' strand—can serve as a template to specify the sequence of nucleotides in a complementary strand. In this way, both strands of a DNA double helix can be copied with precision, producing two exact copies of the original double helix. The chemistry of DNA synthesis. Nucleotides enter the reaction as deoxyribonucleoside triphosphates, and the addition of a deoxyribonucleotide to the 3' end of a polynucleotide chain is the fundamental reaction by which DNA is synthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (the template strand) guides the formation of the new strand of DNA and ensures that its nucleotide sequence is complementary to that of the template. How DNA polymerase adds a deoxyribonucleotide to the end of a growing DNA strand. (A) An incoming deoxynucleoside triphosphate forms a base pair with its partner in the template strand. It is then covalently attached to the free 3' hydroxyl (3' OH) end of the growing DNA strand. The new DNA strand is therefore synthesized in the 5'-to-3' direction. The energy for the polymerization reaction comes from the hydrolysis of a high-energy phosphate bond in the incoming nucleoside triphosphate and the release of pyrophosphate, which is subsequently hydrolyzed to yield two molecules of inorganic phosphate. (B) The reaction is catalyzed by the enzyme DNA polymerase (light green). The polymerase guides the incoming nucleoside triphosphate to the template strand and positions it such that its 5' triphosphate will be able to react with the 3'-hydroxyl group on the newly synthesized strand. How DNA polymerase adds a deoxyribonucleotide to the end of a growing DNA strand. Structure of DNA polymerase, as determined by x- ray crystallography, also showing the replicating DNA. The template strand is the longer, orange strand, and the DNA strand being synthesized is colored red. In each round of DNA replication, each of the two strands of DNA is used as a template for the formation of a new, complementary strand. DNA replication is semiconservative because each daughter DNA double helix is composed of one conserved (old) strand and one newly synthesized strand. Two replication forks moving in opposite directions on the bacteria chromosome, a large circular DNA molecule. Each replication fork has a Y-shaped structure and moves progressively along the DNA, spinning out newly replicated DNA behind it. The stem of the Y is the parent DNA double helix, and the two arms of the Y contain the newly synthesized DNA. Parent DNA strands in orange and newly synthesized DNA strands in red. During its isolation for this experiment, the E. coli DNA folded on itself, accounting for the crossing of the double helix. At each replication fork, the lagging DNA strand is synthesized in pieces. The upper diagram shows two replication forks moving in opposite directions on a double-helical DNA molecule; the lower diagram shows the same two forks a short time later. Because both of the new strands at a replication fork are synthesized in the 5'-to-3' direction, the lagging strand of DNA must be made initially as a series of short DNA strands, which are later joined together. To replicate the lagging strand, the DNA polymerase molecule on that side of the fork uses a backstitching mechanism: it synthesizes a short piece of DNA in the 5'-to-3' direction, stops, and is then moved by its protein machine back toward the fork in order to synthesize the next fragment. During DNA synthesis, DNA polymerase proofreads its own work. If an incorrect nucleotide is accidentally added to a growing strand, the DNA polymerase stops, cleaves it from the strand, and replaces it with the correct nucleotide before continuing. DNA polymerase contains separate sites for DNA synthesis and proofreading. The DNA polymerase, which cradles the DNA molecule being replicated, is shown in the polymerizing mode (left) and in the proofreading, or editing, mode (right). The catalytic sites for the polymerization activity (P) and editing activity (E) are indicated. When the polymerase adds an incorrect nucleotide, the newly synthesized DNA strand (red) transiently unpairs from the template strand (orange), and its 3' end moves into the editing site (E) to allow the incorrect nucleotide to be removed. RNA primers are synthesized by an RNA polymerase called DNA primase, which uses a DNA strand as a template. Like DNA polymerase, primase synthesizes in the 5'-to-3' direction. Unlike DNA polymerase, however, primase can start a new polynucleotide chain by joining together two nucleoside triphosphates without the need for a base-paired 3' end as a starting point. A DNA primase uses ribonucleoside triphosphates rather than deoxyribonucleoside triphosphates, and it is much less accurate than a DNA polymerase. Different enzymes act in series to synthesize DNA on the lagging strand. In eukaryotes, RNA primers are made at intervals of about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. These primers are extended by DNA polymerases at the replication fork to produce Okazaki fragments. The primers are subsequently removed by nucleases that recognize the RNA strand in an RNA–DNA hybrid helix and destroy it; this leaves gaps that are filled in by an accurate “repair” DNA polymerase that proofreads as it fills in the gaps. The completed DNA fragments are finally joined together by an enzyme called DNA ligase, which catalyzes the formation of a phosphodiester bond between the 3'-hydroxyl end of one fragment and the 5'-phosphate end of the next, thus linking up DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis. The ligase enzyme uses a molecule of ATP to activate the 5' phosphate of one fragment before forming a new bond with the 3' hydroxyl of the other fragment How DNA helicase enzymes can separate strands as they move along a DNA single strand. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis. As indicated, many DNA helicases are composed of a ring of six subunits. The structure of a DNA helicase. (A)Diagram of the protein, a hexameric ring, drawn to scale with a replication fork. (B)Detailed structure of the bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, along a DNA single strand that passes through the central hole. Bound ATP molecules in the structure are The effect of single-strand DNA-binding proteins (SSB proteins) on the structure of single- stranded DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The “hairpin helices” result from an intraDNA matching of short regions of complementary nucleotide sequence. Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA), which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein–DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end on. The sliding clamp that holds DNA polymerase on the DNA. The structure of the clamp loader (green) resembles a screw nut, with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits, which enables it to slip around DNA. The loader then “screws” the open clamp onto double-stranded DNA until it encounters the 3' end of a primer, at which point the loader hydrolyzes ATP and releases the clamp, allowing it to close around the DNA. The clamp loader dissociates once the clamp has been assembled. On the lagging strand, it is ready to assemble a new clamp at the start of each new Okazaki fragment. A bacterial replication fork. (A) In this case, a single DNA polymerase molecule synthesizes the leading strand while two DNA polymerases are used—in alternating fashion—for lagging-strand DNA synthesis. All of these polymerase molecules, which are identical, are held in place at the fork by flexible “arms” that extend from the clamp loader. Additional interactions (for example, between the DNA helicase and primase) ensure that all the individual components function together as a well-coordinated protein machine. Schematic diagram of a eukaryotic replication fork. Unlike the bacterial replication proteins, those from eukaryotes are thought to function largely independently, perhaps accounting for the slower speed of the eukaryotic replication fork. Note that the eukaryotic CMG helicase moves unidirectionally along the leading-strand template. DNA duplex is rapidly pried apart at the front of the moving replication fork by harnessing the energy of ATP hydrolysis. CMG (Cdc45-MCM-GINS) https://youtu.be/TNKWgcFPHqw Strand-directed mismatch repair in eukaryotes. (A) The MutS protein binds to a mismatched base pair, recruits the MutL protein, and the complex scans the nearby DNA for a gap and a sliding clamp whose orientation determines which strand is to be cut and its nucleotides replaced. When these are encountered, MutL is activated and begins to cleave the DNA. In most organisms, MutL is joined by another nuclease and, together, they remove the newly synthesized DNA starting at the gap and extending past the mismatch. The gap is then filled in by DNA polymerase d and sealed by DNA ligase. (B) The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS The “winding problem” that arises during DNA replication. (A) For a bacterial replication fork moving at 500 nucleotides per second, the parent DNA helix ahead of the fork must rotate at about 50 revolutions per second. The brackets represent about 20 turns of DNA. (B) If the ends of the DNA double helix remain fixed (or difficult to rotate), tension builds up in front of the replication fork as it becomes overwound. Some of this tension can be taken up by supercoiling, whereby the DNA double helix twists around itself. However, if the tension continues to build up, the replication fork will eventually stop because further unwinding requires more energy than the DNA helicase at the fork can provide. (C) DNA The reversible DNA nicking reaction catalyzed by a DNA topoisomerase I enzyme. As indicated, these enzymes transiently form a single covalent bond with DNA; this allows free rotation of the DNA around the covalent backbone bonds linked to the blue phosphate. On reversal of the reaction, the enzyme and the DNA are restored, the only difference being the relaxation of tension in the DNA. The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze ATP, which is needed to release and reset the enzyme after each cycle. The small yellow circles represent the 59 phosphates in the DNA backbone that become covalently bonded to the topoisomerase. Type II topoisomerases are especially important for rapidly dividing cells. A replication bubble formed by replication-fork initiation. This diagram outlines the major steps in the initiation of replication forks at replication origins. In the last step, two replication forks move away from each other, separated by an expanding replication bubble. DNA replication of a bacterial genome. It takes E. coli about 30 minutes to duplicate its genome of 4.6 x 106 nucleotide pairs. For simplicity, Okazaki fragments are not shown on the lagging strand. The proteins that initiate DNA replication in bacteria. For E. coli DNA replication, the major initiator protein (purple), the helicase (yellow), and the primase (blue) are the dnaA, dnaB, and dnaG proteins, respectively. In the first step, many molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a filamentous structure in which the DNA is wrapped around the protein. Next, two helicases are brought in by helicase- loading proteins (the dnaC proteins; brown), which inhibit the helicases until they are properly loaded at the replication origin. Aided by single-strand binding protein, the loaded helicases further separate the DNA strands, thereby enabling primases to enter and synthesize initial primers. In subsequent steps, two complete replication forks are assembled at the origin and move in Methylation of the E. coli replication origin creates a refractory period for DNA initiation. DNA methylation occurs at GATC sequences, 11 of which are found in the origin of replication (spanning approximately 250 nucleotide pairs). In its hemimethylated state (one strand of the DNA methylated, the other unmethylated), the origin of replication is bound by an inhibitor protein (Seq A), which blocks the ability of the initiator proteins to unwind the origin DNA. About 15 minutes after replication is initiated, the hemimethylated origins become fully methylated by a DNA methylase enzyme; Seq A then dissociates allowing the origin of replication to become active. A single enzyme, the Dam methylase, is responsible for methylating all E. coli GATC sequences. The four successive phases of a standard eukaryotic cell cycle. During the G1, S, and G2 phases, the cell grows continually. During M phase growth stops, the nucleus divides, and the cell divides in two. DNA replication is confined to the part of the cell cycle known as S phase. G1 is the gap between M phase and S phase; G2 is the gap between S phase and M phase. Many eukaryotic cells spend only a small fraction of their time in S phase. The origins of DNA replication on chromosome III of the yeast S. cerevisiae. This chromosome, one of the smallest eukaryotic chromosomes known, carries a total of 180 genes. As indicated, it contains 18 replication origins, although they are used with different frequencies. Those in red are typically used in less than 10% of cell divisions, while those in green are used about 90% of the time. DNA replication initiation in eukaryotes. This mechanism ensures that each origin of replication is activated only once per cell cycle. An origin of replication can be used only if two Mcm helicases are loaded in G1 phase. At the beginning of S phase, specialized kinases phosphorylate both the Mcm helicases and ORC, activating the former and inactivating the latter. These kinases also guide the assembly of additional proteins that complete the helicases to form the fully active replicative helicases, known as the CMG helicases. New Mcm helicases cannot be loaded at the origin until the cell progresses through mitosis to the next G1 phase, when ORC is dephosphorylated. CMG (Cdc45-MCM-GINS) ORC: Origin recognition complex Formation of nucleosomes behind a replication fork. Parent H3–H4 tetramers remain associated with the fork and are distributed at random to the daughter DNA molecules, with roughly equal numbers inherited by each daughter. In contrast, H2A–H2B dimers are released completely from the fork as it passes. This release begins just in front of the replication fork and is facilitated by the histone chaperone FACT. Additional histone chaperones (NAP1 and CAF1) restore the full complement of histones to daughter molecules using both parent and newly synthesized histones. FACT directly transfers parent H3–H4 tetramers to components of the replication machinery, which in turn transfer them to CAF1 chaperones, which deposit them evenly on the two daughter molecules. The way in which histones are distributed behind a replication fork means that some daughter nucleosomes contain only parent Schematic structure of human telomerase. This large enzyme is composed of 10 protein subunits and an RNA of 451 nucleotides. The RNA forms the scaffold of the complex, provides the template for synthesizing new DNA telomere repeats, and helps form the active site. The synthesis reaction itself is carried by the reverse transcriptase domain of the protein, shown in light green, in conjunction with the RNA. A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it. Telomerase also contains several additional protein complexes that are needed to assemble Telomere replication. The 3’ end of the parent lagging-strand template is extended by RNA-templated DNA synthesis; this allows the incomplete daughter DNA strand that is paired with it to be synthesized to the end of the chromosome. The synthesis of the final bit of lagging strand is carried out by DNA polymerase a, which carries a DNA primase as one of its subunits. DNA polymerase a is the same enzyme used to begin the synthesis of each Okazaki fragment on the lagging strand; it begins its synthesis with RNA (not shown) and continues with DNA (green).