Lecture 6 BBT317 Mutation and Repair Part VI.pptx

BBT317 Molecular Genetics Lecture # 6  Mutation and Repair: Part VI Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffith...

BBT317 Molecular Genetics Lecture # 6  Mutation and Repair: Part VI Pierce Genetics: A Conceptual Approach (4e or 5e) Chapter 18 Snustad Principles of Genetics (6e) Chapter 13 Klug Essentials of Genetics (10e) Chapter 14 Watson Molecular Biology of the Gene (7e) Chapter 10 Griffiths Introduction to Genetic Analysis (11e) Chapter 16  DNA Repair Mechanisms Double-strand Break Repair (Griffiths) What would happen if both strands of the double helix were damaged in such a way that complementarity could not be exploited? For example, exposure to X- rays often causes both strands of the double helix to break at sites that are close together. This type of mutation is called a double-strand break. If left unrepaired, double-strand breaks can cause a variety of chromosomal aberrations resulting in cell death or a precancerous state. Interestingly, the generation of double-strand breaks is an integral feature of some normal cellular processes that require DNA rearrangements. One example is meiotic recombination. As will be seen in the remainder of this chapter, the cell uses many of the same proteins and pathways to repair double-strand breaks and to carry out meiotic recombination. Double-strand breaks can arise spontaneously (for example, in response to reactive oxygen species produced as a by-product of cellular metabolism) or they can be induced by ionizing radiation. Two distinct mechanisms are: 1. non-homologous end joining (NHEJ) and 2. homologous recombination. Q1. What are the major pathways to repair DNA double-strand breaks?  DNA Repair Mechanisms Double-strand Break Repair: Non-homologous End Joining (NHEJ) pathway (Literature) NHEJ is an error-prone pathway that repairs double-strand breaks in higher eukaryotes by ligating the free ends back together. NHEJ proceeds through a set of mechanistically distinct steps to join DNA ends (Figure 1). Figure 1: An overview of the NHEJ pathway Upon DSB formation, core and accessory NHEJ factors recognize DNA ends and tether them together in a synaptic complex. If the DNA ends are not compatible for immediate ligation, they are modified by processing enzymes, until ligation can occur. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8899865/ Q1. Discuss the NHEJ pathway to repair DNA double-strand break.  DNA Repair Mechanisms Double-strand Break Repair: Non-homologous End Joining (NHEJ) pathway (Literature) Upon formation of a DSB, the DNA ends are rapidly bound by the Ku70/80 heterodimer, an extremely abundant ring-shaped molecule that tightly encircles DNA. Due to its role as a recruitment hub for many downstream NHEJ factors, Ku binding is a critical DNA end detection step that initiates the assembly of the NHEJ machinery. One downstream NHEJ factor is DNA-PKcs, a large protein kinase belonging to the phosphoinositide 3-kinase (PI3K)-related kinase family. DNA-PKcs recognizes DNA-bound Ku, forming the DNA-PK holoenzyme. DNA binding stimulates DNA-PKcs kinase activity, leading to the phosphorylation of numerous NHEJ and DNA repair factors. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8899865/ Ligation requires close alignment of DNA ends in a synaptic complex. Many NHEJ factors have been implicated in synapsis, including the paralogs XRCC4, XRCC4- like factor (XLF) and Paralog of XRCC4 and XLF (PAXX). Ultimately, DNA ligase IV (LIG4), which forms a constitutive complex with XRCC4, catalyzes ligation. Notably, LIG4 can tolerate certain terminal mismatches and damaged bases, a unique feature among vertebrate ligases. Nonetheless, many DNA end structures are incompatible for direct ligation. Accordingly, a large number of end processing factors including polymerases and nucleases are recruited to DSBs and act on the ends to prepare them for ligation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8899865/ Q1. Discuss the NHEJ pathway to repair DNA double-strand break.  DNA Repair Mechanisms Double-strand Break Repair: Non-homologous End Joining (NHEJ) pathway (Literature) Figure 4: End synapsis during NHEJ DNA ends are rapidly bound by Ku, which recruits further NHEJ factors. Ku, DNA-PKcs, and PAXX enable formation of the long- range synaptic complex, in which the DNA ends are tethered together but separated by >80 Å. Transition to the short-range synaptic complex, in which DNA ends are directly juxtaposed, requires XLF, XRCC4-LIG4, and DNA- PKcs kinase activity. Putative DNA-PKcs autophosphorylation in the short-range complex is shown as orange circles. Here, LIG4 is shown directly engaging both DNA ends to promote short-range https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8899865/ synapsis, but other structural contributions are possible Q1. Discuss the NHEJ pathway to repair DNA double-strand break.  DNA Repair Mechanisms Double-strand Break Repair: Non-homologous End Joining (NHEJ) pathway (Watson) NHEJ is also used in the entirely normal cell-intrinsic process of adaptive immunity. The immune system produces an enormously diverse group of antibody molecules, which are composed of so-called light and heavy polypeptide chains. The light and heavy chains are generated by a recombination process that involves the joining, in a bewildering number of combinations, of a large repertoire of protein-coding DNA elements known as V and J segments (and, in the case of the antibody heavy chain, a D segment) for different parts of the polypeptides. This process is known as V(D)J recombination. V(D)J recombination is initiated by the introduction of breaks in the DNA by a process that is specific to lymphocytes and involves an enzyme composed of the proteins RAG1 and RAG2. Once the breaks are created, the NHEJ pathway, which is not lymphocyte-specific, joins the ends together. In this case, however, the ends of the protein-coding segments are not rejoined to their original partners. Rather, the ends are joined to new partners to create the composite coding sequences for the heavy and light chains. NHEJ also participates in a second example of V(D)J recombination that governs the production of an additional category of immunological polypeptides called T-cell receptors. Q1. Discuss the role of NHEJ pathway in normal cell-intrinsic processes.  DNA Repair Mechanisms Double-strand Break Repair: Non-homologous End Joining (NHEJ) pathway (Watson) Underscoring the importance of NHEJ in human biology are rare inherited syndromes that are characterized by hypersensitivity to ionizing radiation and DNA-damaging agents and by immunodeficiency (SCID), which is attributed to defective V(D)J recombination. Revealingly, patients showing this syndrome harbor mutations in the genes for the Artemis, Ligase IV, or Cernunnos-XLF members of the NHEJ pathway. NHEJ is ubiquitous in eukaryotic organisms, but it occurs, albeit less frequently, in bacteria. Nevertheless, a fascinating specialized example has been discovered in spores of the bacterium Bacillus subtilis. B. subtilis produces a Ku-like protein and a DNA ligase when it sporulates and packages the proteins into the mature spore. Ku and the DNA ligase, representing a simple, two-protein NHEJ system, repair DNA breaks when the spore germinates. Mutant spores lacking these proteins are highly susceptible to dry heat, a condition that is known to cause breaks in DNA. Upon germination, heated mutant spores are unable to resume growth because they are unable to rejoin the heat-induced breaks. Spores of B. subtilis and related bacteria are able to survive extremes of the environment far more effectively than any other kind of dormant cell. NHEJ is part of the basis for this extraordinary robustness. Q1. Why is the NHEJ pathway important? Q2. Is there any example of the presence of NHEJ pathway in bacteria?  DNA Repair Mechanisms Double-strand Break Repair: Homologous Recombination (Griffiths) If a double-strand break occurs after replication of a chromosomal region in a dividing cell, the damage can be corrected by an error-free mechanism called synthesis-dependent strand annealing (SDSA) (homologous recombination). This mechanism is depicted in Figure 16-27. It uses the sister chromatids available in mitosis as the templates to ensure correct repair. Q1. Discuss DSB repair by synthesis-dependent strand annealing (SDSA).  DNA Repair Mechanisms Double-strand Break Repair: Homologous Recombination (Griffiths) The first steps in SDSA are the binding of the broken ends by specialized proteins and enzymes, the trimming of the 5′ ends by an endonuclease to expose single- stranded regions, and the coating of these regions with proteins that include the RecA (E. coli) homolog, Rad51. Rad51 forms long filaments as it associates with the exposed single-stranded region. The Rad51–DNA filament then takes part in a remarkable search of the undamaged sister chromatid for the complementary sequence that will be used as a template for DNA synthesis. This process is called strand invasion. The 3′ end of the invading strand displaces one of the undamaged sister chromatids, which forms a D-loop (for displacement), and primes DNA synthesis from its free 3′ end. New DNA synthesis continues from both 3′ ends until both strands unwind from their templates and anneal. Ligation seals the nicks, leaving a repaired patch of DNA that has one very distinctive feature: it has been replicated by a conservative process. That is, both strands are newly synthesized, which stands in marked contrast to the semiconservative replication of most DNA. Q1. Discuss the role of Rad51 in the DSB repair by homologous recombination.  DNA Repair Mechanisms Double-strand Break Repair: Homologous Recombination (Griffiths) Q1. Discuss the role of Rad51 and other proteins in the DSB repair by homologous https://www.slideshare.net/Raghavv10/homologous-recombination-hr recombination.  DNA Repair Mechanisms Double-strand Break Repair in Meiotic Recombination (Griffiths) Our consideration of the repair of double-strand breaks in dividing cells leads naturally to the topic of crossing over at meiosis because a double-strand break initiates the crossover event. Although the breaks are a normal and essential part of meiosis, they are, if not processed correctly and efficiently, as dangerous as the accidental breaks. Crossing over is a remarkably precise process that takes place between two homologous chromosomes (Figure 16-28). One chromatid from one homologous chromosome will recombine with a non-sister chromatid from the other homologous chromosome. For meiotic segregation to work correctly, every pair of homologs must have at least one crossover. Crossing over is the exchange of genetic material between non- sister chromatids. Q1. What is crossing over?  DNA Repair Mechanisms Double-strand Break Repair in Meiotic Recombination (Griffiths) Meiotic recombination is initiated when an enzyme called Spo11 makes DNA double-strand cuts in one of the chromatids that will recombine (Figure 16-29). Although first discovered in yeast, the Spo11 protein is widely conserved in eukaryotes, indicating that this mechanism to initiate recombination is widely employed. After making its cuts, the Spo11 enzyme remains attached to the now free 5′ ends, where it appears to serve two purposes. First, it protects the ends from further damage, including spurious recombination with other free ends. Second, it may attract other proteins that are needed for the next step in recombination. That step is actually very similar to what happens in the repair of double-strand breaks in dividing cells. The 5′ ends are trimmed back (resected), and a protein complex binds to the single-stranded 3′ ends (see Figure 16-29). That complex includes the Rad51 protein that takes part in that remarkable search for complementarity in the sister chromatid. Q1. Discuss the steps of meiotic recombination.  DNA Repair Mechanisms Double-strand Break Repair in Meiotic Recombination (Griffiths) At this time, meiotic recombination takes a dramatically different path from double-strand-break repair. In meiosis, Rad51 associates with another protein, Dmc1, which is present only during meiosis (see Figure 16-29). Somehow, by an incompletely understood mechanism, the filament containing Rad51–Dmc1 conducts a search for a complementary sequence. However, in contrast with double-strand-break repair, the filament searches a nonsister chromatid from the homologous chromosome, not the sister chromatid. The search culminates in strand invasion and D-loop formation, just as in double-strand-break repair. These events are necessary for chiasma formation in meiosis I. That is, the homologs become connected as a result of recombination. Q1. Discuss the steps of meiotic recombination.  DNA Repair Mechanisms Error-prone Repair: Bypass or Translesion DNA synthesis (Griffiths) A stalled replication fork can initiate a cell-death pathway. In both prokaryotes and eukaryotes, such replication blocks can be bypassed by the insertion of nonspecific bases. In E. coli, this process requires the activation of the SOS system. The name SOS comes from the idea that this system is induced as an emergency response to prevent cell death in the presence of significant DNA damage. As such, SOS induction is a mechanism of last resort, a form of damage tolerance that allows the cell to trade death for a certain level of mutagenesis. Figure 16 -24 A model for translesion synthesis in E. coli. In the course of replication, DNA polymerase III is temporarily replaced by a bypass polymerase (pol V) that can continue replicating past a lesion. Bypass polymerases are error prone. The bacterial β clamp (red protein) is equivalent to the eukaryotic PCNA. Q1. Discuss the steps of translesion DNA synthesis in E. coli.  DNA Repair Mechanisms Error-prone Repair: Bypass or Translesion DNA synthesis (Griffiths) Figure 16-24 shows the steps in the SOS mechanism. In the first step, UV light induces the synthesis of a protein called RecA. RecA is a key player in key mechanisms of DNA repair and recombination. When the replicative polymerase (DNA polymerase III) stalls at a site of DNA damage, the DNA ahead of the polymerase continues to be unwound, exposing regions of single-stranded DNA that become bound by single-strand-binding (SSB) proteins. Next, RecA proteins join the SSB proteins and form a protein–DNA filament. The RecA filament is the biologically active form of this protein. In this situation, RecA acts as a signal that leads to the induction of several genes that are now known to encode members of a newly discovered family of DNA polymerases that can bypass the replication block and are distinct from replicative polymerases. DNA polymerases that can bypass replication stalls have also been found in diverse taxa of eukaryotes ranging from yeast to human. These eukaryotic polymerases contribute to a damage-tolerance mechanism called translesion DNA synthesis that resembles the SOS bypass system in E. coli. Q1. Discuss the steps of translesion DNA synthesis in E. coli.  DNA Repair Mechanisms Error-prone Repair: Bypass or Translesion DNA synthesis (Griffiths) These translesion, or bypass, polymerases, as they have come to be known, differ from the main replicative polymerases in several ways. First, they can tolerate unusually large adducts on the bases. Whereas the replicative polymerase stalls if a base does not fit into an active site, the bypass polymerases have much larger pockets that can accommodate damaged bases. Second, in some situations, the bypass polymerases have a much higher error rate, in part because they lack the 3′-to-5′ proofreading activity of the main replicative polymerases. Third, they can only add a few nucleotides before falling off. This feature is attractive because the main function of an error-prone polymerase is to unblock the replication fork, not to synthesize long stretches of DNA that could contain many mismatches. Q1. What are the major differences between replicative and translesion or bypass polymerases?  DNA Repair Mechanisms Error-prone Repair: Bypass or Translesion DNA synthesis (Griffiths, Literature) Several bypass polymerases that appear to be always present in eukaryotic cells are now known [Pol ζ (zita), Rev1, Pol κ (kappa), Pol η (ita), Pol ι (iota)]. Because they are always present, their access to DNA must be regulated so that they are used only when needed. The cell has evolved a neat solution to this problem. An integral part of the replisome is the PCNA (proliferating cell nuclear antigen) protein that functions as a sliding clamp to orchestrate the myriad events at the replication fork. One critical protein present at a stalled replication fork is Rad6, which, curiously, is an enzyme that adds ubiquitin to proteins (Figure 16-25). The addition of chains of many ubiquitin monomers serves to target a protein for degradation. In contrast, the binding of a single ubiquitin monomer to PCNA changes its conformation so that it can now bind the bypass polymerase and orchestrate translesion synthesis. Enzymatic removal of the ubiquitin tag on PCNA leads to the dissociation of the bypass polymerase and the eventual restoration of normal replication. Any base mismatch due to translesion synthesis still has a chance of detection and correction by the mismatch-repair pathway. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650891/ Q1. What are the eukaryotic bypass polymerases? Q2. What is the significance of monoubiquitination of PCNA with respect to binding with bypass polymerases in eukaryotes?  DNA Repair Mechanisms Error-prone Repair: Bypass or Translesion DNA synthesis (Griffiths) Figure 16 -25 The addition of a single ubiquitin (Ub) monomer to the sliding clamp (PCNA) allows the bypass polymerase to bind to PCNA and begin replicating. Q1. How does ubiquitination regulate binding of PCNA to the bypass polymerase in eukaryotes?  DNA Repair Mechanisms Inherited Human Diseases with Defects in DNA Repair (Pierce) Defects in DNA repair are the underlying cause of several genetic diseases. Many of these diseases are characterized by a predisposition to cancer. Among the best studied of the human DNA-repair diseases is xeroderma pigmentosum (Figure 18.29), a rare autosomal recessive condition that includes abnormal skin pigmentation and acute sensitivity to sunlight. Persons who have this disease also have a strong predisposition to skin cancer, with an incidence ranging from 1000 to 2000 times that found in unaffected people. Sunlight includes a strong UV component; so exposure to sunlight produces pyrimidine dimers in the DNA of skin cells. Although human cells lack photolyase (the enzyme that repairs pyrimidine dimers in bacteria), most pyrimidine dimers in humans can be corrected by nucleotide-excision repair (see Figure 18.28). However, the cells of most people with xeroderma pigmentosum are defective in nucleotide-excision repair, and many of their pyrimidine dimers go uncorrected and may lead to cancer. Q1. What is xeroderma pigmentosum?  DNA Repair Mechanisms Inherited Human Diseases with Defects in DNA Repair (Pierce) Q1. Discuss any seven genetic diseases associated with defects in DNA repair.  Next Lecture: Pedigree Analysis, Applications, and Genetic Testing