DNA_Replication_and_Repair_HO.pdf

DNA Replication and Repair David P. Gardner, Ph.D. M2P Course 2024  Objectives 1. Be able to identify whether nucleotide synthesis is creating DNA (replication) or is creating RNA (transcription). 2. Explain the difficulty associated with...

DNA Replication and Repair David P. Gardner, Ph.D. M2P Course 2024  Objectives 1. Be able to identify whether nucleotide synthesis is creating DNA (replication) or is creating RNA (transcription). 2. Explain the difficulty associated with replicating a linear piece of DNA and explain how this difficulty is overcome in the nucleus. 3. If given a protein involved in DNA replication, be able to restate its function. 4. Diagram and explain the problem with telomere replication and how it is overcome. 5. Speculate why a cell without telomerase activity has a limited capacity for cell division. 6. Compare and contrast DNA damage and mutation. 7. Distinguish spontaneous DNA damage from chemical or radiation types of DNA damage. 8. Generate figures that show the repair of deaminations, depurinations and pyrimidine dimers. 9. Assign a name if provided a description or diagram of each repair mechanism described. 10. Associate Lynch syndrome with mismatch repair and XP with nucleotide excision repair. 11. Illustrate the relationship between MSH and MLH proteins and Lynch syndrome.  Recommended Readings Lippincott® Illustrated Reviews: Cell and Molecular Biology, 3e DNA Replication: Chapter 7 How are 6 Billion Bases Replicated Each Cell Division? In eukaryotes the replication rate is ~50 nucleotides/second. If there was only one origin of replication/chromosome, it would take about a month to replicate all the DNA in a human cell. The task is usually accomplished in ~ 8 hrs. How so much faster? ____________ origins of replication on each chromosome. About 4000 ori/ genome. FIGURE 7.13 Replication origins in eukaryotic chromosomes Molecular Biology of the Cell, 8th Edition  DNA Replication The two strands of the helix must be separated so that the hydrogen bonding donor and acceptor groups become exposed for base pairing. Each nucleotide in the DNA is recognized (through H-bonding) by an unpolymerized free nucleotide. These unpolymerized nucleotides are incorporated into the new strand by a DNA polymerase enzyme.  Prokaryotic DNA Polymerases There are three di erent DNA polymerases in prokaryotic cells and pol III replicates both leading and lagging strands a er primer removal. Polymerase Function Proofreading I Removal and replacement of RNA primer II Involved in replication associated with DNA repair Primary enzyme for leading and lagging strand III synthesis ✔︎ ✔︎ ✔︎ ff ft The Reaction Catalyzed by DNA Polymerase All known DNA polymerases add dNTPs to the 3' hydroxyl group of a growing DNA chain. Note that the energy for DNA replication comes from the incoming nucleotide. Why is this important? FIGURE 7.1 The reaction catalyzed by DNA polymerase Molecular Biology of the Cell, 8th Edition  DNA Polymerase Fidelity A key aspect of high fidelity replication is proofreading. Eukaryotic polymerases δ and ε and bacterial polymerases have a 3’ to 5’ ____________ activity that is opposite of the replication direction. DNA polymerase recognizes mismatches and can back up and remove the base before moving on to incorporating the next nucleotide. This increases fidelity about 1000 fold. Polymerase Function Appears to be primarily involved in δ lagging strand synthesis and DNA FIGURE 7.11 Proofreading repair. Appears to be primarily involved in by DNA polymerase ε Molecular Biology of the Cell, leading strand synthesis 8th Edition  The Di iculty with DNA Replication The two strands of DNA are antiparallel. One is 5' to 3' while the other is 3' to 5'. DNA replication goes in only one direction. For linear DNA this represents a potential problem. FIGURE 7.1 The reaction catalyzed by DNA polymerase Molecular Biology of the Cell, 8th Edition ff  Leading Strand Synthesis The problem is solved by DNA primase, an enzyme that uses ribonucleoside triphosphates to synthesize short RNA primers on the DNA molecule. Unlike a DNA polymerase, DNA primase can initiate RNA strands de novo. Primase synthesizes a short stretch of ______, leaving a 3' hydroxyl group available for DNA polymerase to extend. Eventually, the RNA is removed. DNA primase is a DNA dependent RNA polymerase. It is not a DNA polymerase. 𝝰  Lagging Strand Synthesis Lagging strand synthesis must occur in the 5' to 3' direction. The polymerase must travel away from the advancing replication fork. This seems highly counter intuitive. How can replication of both strands follow DNA unwinding when one of the two strands is replicated in the opposite direction? Mechanism of Lagging Strand Synthesis DNA primase polymerizes an RNA primer on the lagging strand near to where the two strands are initially separated. DNA polymerase (⍺ then δ) can then extend this primer _______ from the replication fork until it runs into the last primer that was created by DNA primase. These short stretches of DNA (1-3 kb) are called Okazaki RNA fragments a er their discoverer. FIGURE 7.4 Initiation of Okazaki fragments with RNA primers Molecular Biology of the Cell, 8th Edition ft  Lagging Strand Synthesis The RNA/DNA duplex is recognized by special repair enzymes and the RNA is removed and replaced with DNA by pol δ. DNA ligase fills in the nicks in the phosphate backbone. Called lagging strand synthesis because it takes longer than leading strand synthesis since there are more steps involved. FIGURE 7.5 Removal of RNA primers and joining of Okazaki fragments Molecular Biology of the Cell, 8th Edition  The Replication Fork The combination of two leading and two lagging strands produces two complete replication forks from one origin of replication.  Proteins at (or near) the Replication Fork DNA Helicase SSB Proteins Sliding Clamp Clamp Loading Protein Protein DNA Primase DNA polymerase Topoisomerase I and II* *Topoisomerases not shown in this image. If given a protein involved in DNA replication, be able to restate its function.  The Replisome You are not required to know the 3 dimensional complexity of this video. 2nd useful video link. FIGURE 7.10 Model of the E. coli replication fork Molecular Biology of the Cell, 8th Edition  Telomere Replication Telomeres are the _______ of linear chromosomes. They consist of a simple repeat sequence present ~2500 times per telomere. Chromosome ends present unique problems with respect to replication. Note that the very end of one of the two strands of the DNA molecule cannot be replicated by DNA polymerase. The overhang would be removed resulting in a shorter chromosome with DNA synthesis + each replication. RNA removal Telomere Replication Telomerase enzyme consists of a reverse transcriptase plus a unique _____. The RNA hybridizes to the overhang on the DNA. This RNA provides a template for extension of the upper strand by telomerase reverse transcriptase activity. FIGURE 7.16 Action of telomerase Molecular Biology of the Cell, 8th Edition  Telomere Replication Process can be repeated multiple times resulting in an extension of the overhang. An RNA primer can hybridize to the very end and fill in the gap. Removal of RNA still leaves a gap but as long as multiple repeats are added, no sequence is lost. Note this solution simply avoids the fundamental FIGURE 7.16 Action of telomerase Molecular Biology of the Cell, 8th Edition problem of replicating the end of the DNA molecule.  Telomeres in Somatic Cells Most cells in the body lack telomerase activity. A critical exception being stem cells. Telomeres in somatic cells get shorter with each cell division. Chromosomes lacking telomeres is a signal for apoptosis. The absence of telomerase limits the number of cell divisions possible for a somatic cell to about 50. Human lymphocyte chromosomes hybridized with fluorescent labeled What about cancer cells? TTAGGG sequence.  Importance of DNA Repair It is estimated that with every round of DNA replication, 100-200 new mutations are introduced into the human genome. A lot of cellular resources are utilized to replicate DNA correctly and detect and correct errors that do occur. Inability to correct DNA damage results in mutations. DNA Damage ≠ Mutations. Mutations cannot be repaired. DNA damage can. New mutations are a driving force of many genetic diseases.  DNA Damage DNA damage is an alteration in the DNA of a cell that, if le uncorrected, would result in a mutation in that cell or possibly the death of the cell. In contrast to mutations, which occur rarely, DNA damage occurs with very high frequency. Approximately 99.98% of all DNA damage is repaired. ft  Types of DNA Damage DNA, like any other molecule, can undergo chemical reactions. These reactions can occur spontaneously or as a result of exposure to chemicals or radiation. Such reactions would result in an inability to continue DNA replication and/or transcription of genes. In addition, uncorrected DNA damage greatly increases the mutation frequency. Any of these would be bad.  Spontaneous DNA Damage There are two major types of spontaneous DNA damage. The first is ______________. Deamination of DNA is the loss of an amine group from adenine, cytosine and guanine. (Why not thymine?) Deamination of cytosine creates uracil-normally found only in RNA. Deamination of adenine creates hypoxanthine: a base not found in DNA. Deamination of guanine creates xanthine, also not found in DNA. Note that deamination never generates a base normally found in DNA.  Deaminations Uracil resembles thymine and hydrogen bonds with adenine, thus a C-G pair would be changed to an A-T pair by the uncorrected deamination of cytosine. Why did the genetic code evolve to avoid the use of uracil, xanthine and hypoxanthine as normal bases in DNA?  Depurinations The 2nd type of spontaneous damage is depurination. This is like ge ing your head cut o. The glycosidic bond between the purine base and the deoxyribose sugar is cleaved leaving an apurinic (AP) site. Depyrimidinations also occur but are less common. tt ff  Radiation and Chemical Damage UV light can induce formation of pyrimidine dimers. Pyrimidine dimers form when two adjacent pyrimidines are joined by a cyclobutane ring structure. FIGURE 7.17 Examples of DNA damage Molecular Biology of the Cell, 8th Edition  Mechanisms for Repair of DNA Two Main Categories: 1. Direct reversal of the chemical reaction responsible for the damage. 2. Removal of the damaged base or bases, o en along with flanking DNA. Followed by replacement. Most damage in human cells is repaired by the second method, called excision repair. Excision repair comes in two primary flavors: Base excision repair Nucleotide excision repair ft  Base Excision Repair Base excision repair results in the removal of a ________ damaged base. Step 1 involves enzymes called DNA glycosylases. DNA glycosylase recognizes specific altered bases and catalyzes their removal. Multiple DNA glycosylases exist, each with a di erent specificity for damage. FIGURE 5.19 Base-excision repair Molecular Biology of the Cell, 9th Edition ff Repair A er Uracil Incorporation Repair begins with DNA glycosylase recognizing and removing the incorrect base (uracil in this example). The enzyme cleaves the glycosidic bond between the base and the DNA backbone. This creates an AP site that is recognized by an AP endonuclease that cleaves the DNA backbone. ft  Repair A er Uracil Incorporation A er the AP endonuclease cleaves the backbone, the remaining deoxyribose sugar is removed by deoxyribosephosphodiesterase. This produces a 3’ _____ to extend. The gap in the strand is filled in by DNA polymerase β. Finally, the correctly incorporated base is sealed into the strand by DNA ligase. ft ft  Repair of Depurination Depurinations are also corrected by base excision repair. Spontaneous depurination creates an AP site. AP endonuclease recognizes the AP site. Same as repair of uracil except that a glycosylase enzyme is not needed.  Nucleotide Excision Repair A less specific repair mechanism. It can repair any damage that alters the _______________ of the DNA. Typically the addition of bulky adducts to the bases (includes pyrimidine dimers). System relies upon the fact that this type of damage alters the normal shape of the double helix. First step in the process is looking for alterations in the shape of the helix. Nucleotide Excision Repair Overview Damage is recognized (shape change). Damaged strand is cleaved upstream and downstream of the damage. Helicase unwinds the two strands. Gap is filled in by a DNA polymerase. Ligase seals the gap. FIGURE 5.20 Nucleotide-excision repair of thymine dimers The Cell, A Molecular Approach, 9e Nucleotide Excision Repair Altered bases are recognized by a protein called XPC. Recruited to the complex next are XPA, RPA (a SSBP), and the general transcription factor TFIIH. XPA seems to function to properly organize the proteins that bind the damaged site. XPD and XPB are subunits of TFIIH and are helicases that unwind about 30 bp of DNA surrounding the damage. Next, XPG plus a dimer of XPF and ERCC1 proteins bind the site. FIGURE 5.21 Nucleotide-excision repair The Cell, A Molecular Approach, 9e Nucleotide Excision Repair XPG and XPF/ERCC1 are endonucleases that cleave the damaged strand 5’ and 3’ of the damage. Approximately 30 bp surrounding the damage are removed. DNA polymerase δ plus RFC and sliding clamp recognizes the 3’-OH and fills in the gap. DNA ligase links the new DNA with the old  Xeroderma Pigmentosum What goes wrong in xeroderma pigmentosum? Faulty mechanism. Genes, Proteins, Mode of Inheritance, Clinical Presentation. Frequency? Good site for this info? Diseases Caused by Defective DNA Repair  Mismatch Repair Genes involved in the mismatch repair process are commonly mutated in Lynch syndrome. A major, but not only, phenotype of Lynch syndrome is colorectal cancer (O en called HNPCC: hereditary nonpolyposis colorectal cancer). Lynch syndrome is autosomal dominant and accounts for ~15% of all colorectal cancer cases. What is microsatellite instability? ft  Mismatch Repair Mismatch repair removes incorrect (but normal) bases that have slipped by the proofreading activity of DNA polymerase. How do you know which base is the incorrect base? System can identify newly replicated DNA from the original template DNA. Data suggests it is nicking of the newly made strand in eukaryotes. The Cell, A Molecular Approach, 9e In bacteria? Fig. 5.23  Mismatch Repair in Bacteria In bacteria, the system recognizes ____________ of the old strand at GATC sequences. MutS recognizes the mismatch. MutL then binds. MutL activates MutH A unique enzyme to bacteria, MutH, cleaves the new strand at the region of methylation. MutS and MutL plus a helices and exonuclease (not shown) remove the new DNA including the region of mismatch. Mismatch Repair, Eukaryotes Process begins with ______ protein binding to a site of mismatch. MLH then binds and the two proteins coordinate removal of DNA (5’ → 3’) from the new strand until a nick is reached. On the lagging strand, removal is to the next Okazaki fragment. On the leading strand it is the growing 3’ end. MSH= MutS homolog MLH= MutL homolog, there is no MutH homolog. FIGURE 5.23 Mismatch repair The Cell, A Molecular Approach, 9e  Lynch Syndrome MSH and MLH proteins are coded by multiple genes in humans. ~50% of Lynch syndrome cases are caused by mutation of the MLH1 gene. ~40% of all cases of are due to mutation in the MSH2 gene. 7-10% by mutation of the MSH6 gene. PMS2 gene function?