Textbook Assignment: Chapter 14, pages 406-427 (Pages 416 through 421 are not covered in detail in this course).
Major concepts
Frameshifts and deletions in human mutations: The textbook emphasizes the role played by frameshifts and deletions in two classes of human genetic systems. One is the ABO blood type locus. We saw in chapter 4 (figure 4.2) that the A and B blood groups were determined by glycosyl transferase activities that made two slightly different modifications to the carbohydrate side chain of H substance, adding either an acetylgalactosamine (type A) or a galactose (type B) to the precursor. A consistent set of four single base substitutions was found between the DNAs coding for the A and B types. Type O results from total absence of the glycosyl transferase activity. This has been shown to be caused by a deletion leading to a frameshift early in the coding sequence for the enzyme. Thus, although type O is a common allele, it appears to have been caused by an ancestral frameshift mutation that somehow became well established in the overall population.
Duchenne and Becker Muscular dystrophies: Duchenne and Becker muscular dystrophies both result from defects in a huge protein (3,865 amino acids in length) called dystrophin . The more severe Duchenne Muscular Dystrophy (DMD) is associated with complete absence of a funcitonal protein, and in most cases arises from major deletions or insertions in in the dystrophin gene, which is over 2 megabases in length. The much milder Becker muscular dystrophy (BMD) generally has detectable dystrophin protein that has been rendered less than fully functional by more modest genetic changes. From these observations, the textbook authors generalize that single nucleotide substitutions are far more likely to be tolerated that more extensive genetic changes.
Triplet repeats: The textbook inserts a short discussion of triplet repeats into the current chapter on mutations. This adds a few more triplet repeat diseases, but does not expand significantly on the previously discussed concept that an excess of triplet repeats leads to pathology, which tends to be more severe and have an earlier onset as the number of triplet repeats becomes more excessive.
Thymine dimers: Ultraviolet radiation causes a dimerization reaction between adjacent thymine residues in DNA (fig. 14.14) that disrupts mormal base pairing and functioning of that immediate region of the DNA molecule. A variety of mechanisms exist to reverse such damage in all types of cells ranging from bacteria to higher eukaryotes. Many of the same mechanisms are also used to repair other types of DNA damage that lead to mismatches of base pairing or single-stranded gaps. One of the repair mechanisms is the so-called SOS response, discussed later in this lecture, which results in error-prone repair and thus produces mutations.
Photoreactivation: The first line of defense against uv damage in bacterial cells is photoreactivation. In E. coli an enzyme produced from the phr locus, called photolyase (in our textbook called photoreactivation enzyme, PRE), binds to the thymine dimers in the dark and then uses visible light energy to break the dimer bonds, restoring the original sequence of normal bases (TT). This directly reverses the dimerization process without any need for excision of the damage and replacement of the missing bases. Similar photoreactivation enzymes have been widely conserved across the evolution of highly diverse species.
Excision repair systems: In cell strains that lack the photoreactivation system, and in other cases where it has failed to repair the damage, a variety of excision repair systems can be called into action. Such systems remove the immediate damage and some amount of the strand that contained it, followed by synthesis of a new strand, using the undamaged partner as a template.
Excision of ultraviolet damage: In E. coli , a special endonuclease consisting of three subunits produced from the uvr-A, uvr-B, and uvr-C genes nicks the damaged strand at specific locations upstream and downstream from a thymine dimer. This allows a helicase enzyme to remove a segment of 12 bases including the dimer from the damaged strand, leaving a gap that is then filled in by DNA polymerase I and DNA ligase. In eukaryotic cells, a sequence of 28 bases is removed and replaced, rather than the 12 bases removed and replaced in prokaryotes.
AP repair: Enzymes known as DNA glycolyases are capable of removing damaged bases from DNA by hydrolyzing the bond between the base and the 1'-position of deoxyribose. This leaves apurinic or apyrimidinic sites in the double stranded DNA. Specific AP endonucleases then nick the damaged strand upstream and downstream from the AP site, resulting in removal of a short segment, which is then replaced by DNA polymerase I and DNA ligase in a manner similar to excision repair.
Proofreading and mismatch repair: When a mismatch is detected in a newly synthesized segment of DNA, a mismatch repair system is called into action. The first problem is for the system to decide which is the parental (correct) strand and which is the newly synthesized (presumably error-containing) strand. Methylation of the adenine in GATC sequences serves temporarily as a marker for the parental strand in newly synthesized DNA in E. coli. GATC forms a pallindromic sequence in double stranded DNA (one that reads the same in either direction). If one starts with GATC methylated in both strands, the newly synthesized complementary strands will temporarily lack methylation. GATC sequences with one adenine methylated and the other not are recognized by an enzyme known as deoxyadenosine methylase (dam), which methylates the new strand. However, there is a short time lag after replication before this occurs.
Mismatch detection: Four proteins, coded by genes designated mutH, mutL, mutS, and mutU, participate in mismatch repair. The S and L proteins are involved in recognition of the mismatch (which distorts the normal double helical structure). The H protein recognizes the newly synthesized strand (not methylated) and nicks it. The U protein helps to open the nicked area, which is then stabilized in the open configuration by single-stranded DNA-binding protein. DNA polymerase I and ligase then make the repair, using the methylated strand as a template. The whole process requires about three minutes in E. coli. This proofreading system catches errors missed by the proofreading function of DNA polymerase III, and is believed to improve fidelity of DNA synthesis by about two orders of magnitude (100 fold).
Postreplicative repair: If damage such as a thymine dimer is not detected and removed, it will cause problems during the next round of DNA synthesis that can result in its repair. When DNA polymerase III encounters a thymine dimer, it temporarily stops synthesis, leaving a gap in the newly synthesized strand, which is started again further along the template strand that contains the dimer. This gap triggers a process known as postreplicative repair or recombinational repair. A protein known as the RecA protein (coded at the recA locus) coats the single stranded DNA (the template strand that failed to make a partner because it contains a thymine dimer). This coated strand then invades the newly replicated double-stranded DNA that has just been synthesized from the other template strand at the same replication fork, and causes the damaged strand (with the dimer) to pair with the complementary undamaged template strand. This leaves the newly synthesized undamaged strand unpaired, which is not a problem because it is a correct template for repair enzymes to replace the strand that has been taken from it and paired with the damaged strand. The damaged strand and its newly found undamaged partner now have another chance for any of the possible repair mechanisms to remove the thymine dimer before the next round of replication. Because this repair system responds to a signal of distress (DNA damage), it is sometimes referred to as the SOS response .
The SOS response: In addition to the recombinational repair described above, interaction of the recA protein with single stranded DNA (indicative of genetic damage the cell is attempting to repair) causes the recA protein to gain the ability to inactivate a regulatory protein known as LexA. The normal function of the LexA protein is to repress expression of about 20 genes that are involved in various types of emergency repair processes. These include some genes whose products stop the cell cycle, giving the cell more time to achieve appropriate repairs when an emergency is declared (by the absence of enough LexA to keep the emergency response proteins turned off). Each of the genes that is repressed by LexA has a consensus sequence in its promoter, called the SOS box (5'-CTGX10CAG-3') that serves as a binding site for the LexA protein. Among the genes that are activated are those involved in excision of thymine dimers, as well as a variety of others.
Xeroderma pigmentosum: Xeroderma pigmentosum is a human genetic disease (or more correctly, a family of closely related genetic diseases), in which there is abnormal sensitivity to ultraviolet radiation. A number of different genes appear to be involved. Some patients exhibit defects in photoreactivation, but loss of excision repair is more common. Complementation testing done by fusing cultured cells from different patients with defects in excision repair has revealed the presence of at least seven different complementation groups, suggesting that defects in at least seven different loci that code for proteins involved in excision repair can cause the symptoms associated with Xeroderma pigmentosum.
Ionizing radiation: High energy radiation, such as X-rays and gamma rays, causes ionization in the substances that it passes through. The highly reactive free radicals that are generated cause a variety of types of biological damage, including mutation. Breakage of chromosomes is a common result of such irradiation, leading to a variety of deletions, translocations and other aneuploidies. Many early genetic studies on Drosophila used X-rays to incuce mutations, which frequently produced cytological alterations in polytene chromosomes that helped to localize the mutational changes to particular chromosomal areas.
Site-directed mutagenesis: One of the more interesting examples of how cloned genes can be manipulated is site-directed mutagenesis, which our textbook has chosen to describe in this chapter prior to the discussion of gene cloning. I have chosen to delay discussion of this topic until the following lecture, which describes both gene cloning and the single-stranded M13 vector that is used for site-directed mutagenesis.
Knockout organisms: This in one of several procedures that allow non-functional genes to be created and ultimately inserted into the genomes of living species, sometimes completely displacing the funcitonal genes to generate knockout organisms. The discussion in this chapter is rather sketchy and somewhat premature. The generation of knockout organisms is explored more thoroughly in chapter 16 (pages 469-471).
Transposable genetic elements: The final part of Chapter 14 is devoted to a discussion of transposable genetic elements. These are genetic elements that can "jump" from one location to another within a genome, sometimes carrying specific genetic information with them, and sometimes disrupting the function of genes that they land in the middle of. This topic is explored in detail in MCDB 3500, and will not be discussed in this class beyond a minimal level to make you aware of the phenomena. One interesting footnote is that the wrinkled trait in garden peas is caused by the insertion of a transposable element into the structural gene for a starch branching enzyme, thereby disruptong its function (figure 14.25).
You are encouraged to read pages 416 through 421, but you will not be held responsible for learning the material in detail.