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Week 4 Textbook Notes

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Melody Neumann

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BIO130 – Week 4 Pgs: bottom of 276-281, 292-304, 329-339 DNA Replication, Repair and Recombination A Strand-Directed Mismatch Repair System Removes Replication Errors that Escape from the Replication Machine • The strand-directed mismatch repair system detects the potential for distortion in the DNA helix from the misfit between noncomplementary base pairs • In order to be effective, a correction mechanism must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred. Otherwise the parental template strand would be erroneously changed for all further replications • Methyl groups are added to all A residues in the sequence GATC, but not until sometime after the A has been incorporated into a newly synthesized DNA chain o The only GATC sequences that have not yet been methylated are in the new strands just behind the replication fork o Recognition of these unmethylated GATCs allow the new DNA strands to be transiently distinguished from old ones, as required if their mismatches are to be selectively removed o The process has three steps:  (1) the recognition of a mismatch  (2) excision of the segment of DNA containing the mismatch from the newly synthesized DNA  (3) resynthesis of the excised segment using the old strand as a template • Eukaryotes do not rely on methylation to distinguish between strands o Newly synthesized lagging-strand DNA transiently contains nicks and such nicks, (also called single-strand breaks) provide the signal that directs the mismatch proofreading system to the appropriate strand DNA Topoisomerases Prevent DNA Tangling During Replication • The “winding problem” is the fact that every 10 base pairs corresponds to one complete turn about the axis of the parental double helix o Therefore, for a replication fork to move, the entire chromosome ahead would normally have to rotate rapidly – this would be inefficient • DNA topoisomerase is a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand BIO130 – Week 4 o This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves • Topoisomerase I produces a transient single-strand break, (or nick); this break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point • Topoisomerase II forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand break in the helix o These enzymes are activated by sites on chromosomes where two double helices cross over each other o Once topoisomerase II binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently:  (1) it breaks one double helix reversibly to create a DNA “gate”  (2) it causes the second, nearby double helix to pass through this break  (3) it then reseals the break and dissociates from the DNA o Using this method, topoisomerase II can efficiently separate two interlocked DNA circles Telomerase Replicates the Ends of Chromosomes • When the replication fork meets its end, there is no place to produce the RNA primer needed to start the last Okazaki fragment at the very tip of a linear DNA molecule • Eukaryotes have specialized nucleotide sequences at the ends of their chromosomes that are incorporated into structures called telomeres • Telomeres contain many tandem repeats of a short sequence, (in humans it is GGGTTA), that is repeated roughly a thousand times at each telomere • Telomere DNA sequences are recognized by sequence-specific DNA-binding proteins that attract an enzyme, telomerase, that replenishes these sequences each time a cell divides • Telomerase recognizes the tip of an existing telomere DNA repeat sequence and elongates it in the 5’-to-3’ direction, using an RNA template that is a component of the enzyme itself to synthesize new copies of the repeat o Once the parental DNA has been extended by telomerase, replication of the lagging strand at the chromosome end can be completed by the conventional BIO130 – Week 4 DNA polymerases, using these extensions as a template to synthesize the complementary strand Telomere Length is Regulated by Cells and Organisms • In somatic cells of humans, the telomere repeats have been proposed to provide each cell with a counting mechanism that helps prevent the unlimited proliferation of wayward cell in adult tissues o Stem cells retain full telomerase activity, other cells can lose between 100-200 nucleotides from each telomere every time they divide o Replicative cell senescence occurs when the cells begin to inherit defective chromosomes, (because their tips cannot be replicated), and withdraw from the cell cycle and stop dividing DNA Repair Without DNA Repair, Spontaneous DNA Damage Would Rapidly Change DNA Sequences • DNA of each human cells loses about 5000 purine bases, (A and G) every day because their N-gylcosyl linkages to deoxyribose hydrolyze, a reaction called depurination • A spontaneous deamination of C to U in DNA occurs at a rate of about 100 bases per cell per day • DNA bases are also damaged by encounters with reactive metabolites produced in the cell, or by exposure to chemicals in the environment • UV radiation from the sun can produce covalent linkages between two adjacent pyrimidine bases in DNA to form, thymine dimers for example DNA Damage can be Removed by more than one Pathway • Base excision repair involes a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal o These enzymes travel along DNA to evaluate the status of each base, once the enzyme finds the damaged base that it recognizes, it removes the base from its sugar o This now missing base is recognized by an enzyme called AP endonuclease which cuts the phosphodiester backbone, after which the damage is removed and the resulting gap repaired • Nucleotide excision repair can repair the damage cause by almost any large change in the structure of the DNA double helix BIO130 – Week 4 o Errors include those created by the covalent reaction of DNA bases with large hydrocarbons, as well as various pyrimidine dimers caused by sunlight o A large multienzyme complex scans the DNA for a distortion in the double helix, rather than a specific base change o Once a bulky lesion is found, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away a single-strand oligonucleotide containing the lesion o The gap is repaired by DNA polymerase and DNA ligase Coupling DNA Repair to Transcription Ensures that the Cell’s Most Important DNA is Efficiently Repaired • Cells have a way of directing DNA repair to the DNA sequences that are most urgently needed • RNA polymerase is linked to the repair of DNA damage • RNA polymerase stalls at DNA lesions and, through the use of coupling proteins, directs the repair machinery to these sites o The RNA polymerase is backed up, damage is repaired and the polymerase is restarted The Chemistry of the DNA Bases Facilitates Damage Detection • Every possible deamination event in DNA yields an “unnatural” base, which can be directly recognized and removed by a specific DNA gylcosylase • Spontaneous deamination of C converts it to U. If DNA had U as a natural base, the repair system would find it hard to distinguish between a deaminated C and a naturally occurring U, this is why even though RNA was around before DNA, DNA uses T instead of U • In inactive areas C-G pairs are methylated, and deamination of one of those Cs turns it into a T. The repair mechanism at this site is ineffective at replacing the new T to an original C as mutations in these areas account for about one third of the single-base mutations that have been observed in human diseases Double-Strand Breaks are Efficiently Repaired • An especially dangerous type of DNA damage occurs when both strands of the helix are broken, leaving no intact template strand to enable accurate repair o If unrepaired, these breaks could lead to the breakdown of chromosomes into smaller fragments and the loss of genes when the cell divides BIO130 – Week 4 • Nonhomologous end-joining, is an end-joining mechanism in which the broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of one of more nucleotides at the site of joining o So little of the mammalian genome codes for proteins that this mechanism is apparently acceptable as a solution despite it resulting in loss of information • Homologous recombination is much more accurate and occurs in newly replicated DNA o DNA is repaired using the sister chromatid as a template DNA Damage Delays Progression of the Cell Cycle • Eukaryotic cells delay progression of the cell cycle until DNA repair has been completed • The presence of DNA damage can block the transition of a cell from one phase to the next of the cell cycle • DNA damage results in an increase synthesis of some DNA repair enzymes How Cells Read the Genome: From DNA to Protein Transcription Produces RNA Complementary to One Strand of DNA • The enzymes that perform transcription are called RNA polymerases • RNA polymerases catalyze the formation of phosphodiester bonds that link the nucleotides together to form a linear chain • RNA chain is extended in the 5’-to-3’ direction • The hydrolysis of high-energy bonds, (triphosphates on nucleotides), drive the reaction forward as in DNA replication • RNA strand is almost immediately released from the template DNA strand as its synthesized and thus many copies of RNA can be made from the same gene in a relatively short time, with the newest RNA being made before the first has finished • Unlike DNA polymerase, RNA polymerase can start an RNA chain without a primer o RNA polymerase makes far more mistakes than does DNA polymerase • Despite similar functions, DNA and RNA polymerases are structurally very dissimilar. It seems like these two polymerase enzymes have arisen independently Cells Produce Several Types of RNA BIO130 – Week 4 • The RNA molecules that lead to proteins are called messenger RNA, (mRNA) • Many genes end product is RNA rather than proteins o These RNA can serve as enzymatic and structural components o Small nuclear RNA (snRNA) direct the splicing of pre-mRNA to form mRNA o Ribosomal RNA (rRNA) form the core of ribosomes o Transfer RNA (tRNA) form the adaptors that select amino acids and hold them in place on a ribosome for the incorporation into protein o MicroRNA (miRNA) and small interfering RNA (siRNA) molecules serve as key regulators of eukaryotic gene expression • Each transcribed segment of DNA is called a transcription unit o Transcription unit usually carries the information for just one gene, and therefore codes for a single RNA molecule or a single protein o In bacteria, a set of adjacent genes is often transcribed as a unit • RNA only makes up a few percent of a cell’s dry weight o Most of the RNA in cells is rRNA; mRNA comprises only about 3-5% of total RNA in a typically mammalian cell Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop • In bacteria: • A detachable subunit called sigma factor associate with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing o Together the sigma factor and core enzyme are known as the RNA polymerase holoenzyme  Holoenzyme only weakly adheres to bacterial DNA and slides rapidly along the DNA molecule before dissociating • When the polymerase holoenzyme slides into a region on the double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, the polymerase binds tightly to this DNA o Then it opens the double helix to expose a short stretch of nucleotides on each strand BIO130 – Week 4  This does NOT require ATP o After the first ten or so nucleotides have been synthesized, the core enzyme breaks its interactions with the promoter DNA, weakens its interactions with the sigma factor, and begins to move down the DNA synthesizing RNA o Elongation continues until it reaches a second signal, the terminator, where the polymerase halts and releases both newly made RNA chain and the DNA template o Once released, the RNA polymerase reassociates with a free sigma factor to form a holoenzyme that can begin the process of transcription again • Termination signal consists of a string of A-T pairs preceded by a two-fold symmetric DNA sequence, which when transcribed into RNA, folds into a “hairpin” structure o As the polymerase transcribes across a terminator, the formation of the hairpin helps to pull the RNA transcript from the active site, (active site at terminator is largely A and U which is less stable than G-C pairing so pulls out slightly easier) Transcription Start and Stop Signals Are Heterogeneous in Nucleotide Sequence • A consensus nucleotide sequence is derived by comparing many sequences with the same basic function and tallying up the most common nucleotide found at each position • Promoters vary widely in their strength, usually corresponding to their use. If a protein is used in abundance, its promoter sequence is much stronger than a protein used rarely Transcription Initiation in Eukaryotes Requires Many Proteins • Eukaryotes have three types of RNA polymerase: RNA polymerase I, II, and III each transcribing different types of genes • RNA polymerase I and II transcribe genes encoding for transfer RNA, ribosomal RNA, and various small RNAs • RNA polymerase II transcribes most genes, including all those that encode proteins • Difference
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