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DNA Repair and Recombination Mutations • A mutation is any permanent change in the DNA nucleotide sequence. • Simplest form of mutation: a nucleotide substitution, where one nucleotide is replaced by a different one. • Many mutations occur in non-gene areas of the DNA and have very little or no effect on the organism. • Only about 1.5% of human DNA is contained in genes. • In bacteria, 80% or more of the DNA is in genes. • Mutations in genes can alter or destroy the function of the gene product. • A closely related issue: DNA damage, which can stop DNA replication or cell division • DNA polymerase may not be able to use a non-standard nucleotide as a template. • Single stranded breaks in the DNA molecule will have trouble during DNA replication • Double-stranded breaks are worse: the two ends of the broken DNA need to be re-assembled before cell division or replication can succeed. • There are several mechanisms the cell uses to detect and repair DNA damage before it become permanent. Somatic vs. Germ Line Mutations • An important distinction: germ line mutations vs. somatic mutations. • germ line mutations occur in the cells that become sperm or eggs (= gametes). Germ line mutations heritable: they can be passed on to future generations. • On the other hand, somatic mutations occur in other body cells, and can only affect the individual, not any descendants. • Most somatic mutations have little effect on the individual. However, cancer is caused by certain specific somatic mutations. • This distinction mostly applies to animals. In the early embryo, animals set aside a small group of cells to become the germ line. • In plants, any cell has a chance of becoming a gamete. • Single cell organisms don’t use a germ line either. Mutations and Evolution • Mutations are the raw material of evolution. Species change over time due to the effects of natural selection on organisms with different mutations. • Most mutations either have no effect or a deleterious effect on the fitness of an organism (fitness = ability to survive and reproduce). • Occasionally a mutation increases an organism’s fitness. Natural selection causes the frequency of these genetic alleles in the population. • Sometimes a mutation has no effect on fitness, or even a slightly bad effect, but a change in the environment makes the mutation valuable. This is called a preadaptive mutation or exaptation. • Mutations can cause a lot of harm to an organism. Thus it is important to minimize the number of mutations. It is not possible to prevent them, but it is possible to repair the damage they cause. The chart above shows the effect of various mutations on vesicular stomatitis virus. The unmutated virus has a fitness of 1.0. Almost half of the mutations give a fitness of 0 (= dead), and most of them are deleterious (fitness less than 1.0). A few mutations have a fitness greater than 1 (i.e., they are better than the original). Mutations vs. DNA Damage • The mutation process starts with DNA damage (or replication errors), but only becomes an actual mutation if the damage is not repaired. • Initially, the damage or mis-incorporated base causes a mispairing in the DNA double helix: there is something other than an A-T or G-C base pair. • Many of these mispairings are identified and repaired by the cell. There are enzymes that detect any spots on the DNA that are not AT or G-C base pairs. • The damage becomes a mutation only after the mispaired bases are replicated. This converts the mispaired bases into regular A-T and G-C base pairs that look completely normal to the cell’s enzymes. • The mutant bases are now permanently part of the DNA, inherited in all future cell generations. Sources and Types of Mutation • Internal sources: • sometimes DNA polymerase makes a mistake, despite the proofreading function • Interactions between DNA and other molecules in the cell, including water and reactive oxygen species. • External sources • Ultraviolet light and other ionizing radiation • Reactive chemical compounds • Changes in a single nucleotide • Breaking the DNA backbone, either a single stranded break or a double stranded. DNA Polymerase Errors • As we discussed in the Replication lecture, DNA polymerase sometimes incorporates the wrong nucleotide. • Often due to the tautomeric shift, where movement of a Hydrogen atom puts the base in an unusual configuration that allows mispairing. • DNA polymerase detects many of these errors as soon as they occur. • At which point the DNA polymerase uses its 3’ → 5’ exonuclease activity to remove the new base and try again. • The process of detected and correcting these mismatches is called proofreading. • Some errors still occur, and need to be corrected. Spontaneous Depurination and Deamination • Some DNA damage is simply the result of the aqueous environment. H2O spontaneously dissociates into H+ and OH-, which are reactive. • The bond between a purine base and deoxyribose is prone to being hydrolyzed by H+ ions. This is depurination, and it leads to an apurinic site. DNA polymerase usually skips over the apurinic site, producing a 1 base pair deletion in the daughter DNA molecule. • The amino group on the cytosine base is also easily lost by hydrolysis. This is deamination, and it converts cytosine into uracil. Uracil pairs with adenosine, so after replication, a C-G base pair has been converted to an A-T base pair. • Note that uracil is not a DNA base. • Amino groups on other bases (e.g. guanine) can also be lost, converting the base to something non-standard. Oxidative Damage and Alkylating Agents • Aerobic metabolism in the mitochondria uses oxygen. Some of this oxygen gets converted to reactive oxygen species, primarily hydrogen peroxide (H2O2) and superoxide (O2-) • Reactive oxygen species can attack bases, as well as break the DNA backbone. • They also cause other forms of damage in the cell. • Many chemical compounds can attack various bases and attach bulky alkyl (hydrocarbon) groups. • DNA polymerase has a hard time processing these modified bases. • Tobacco smoke contains benzo(a)pyrene, which is modified by the liver to produce a reactive derivative. • This derivative attacks purines and adds a bulky group that does not form proper base pairs. UV-Induced Thymine Dimers • Two T’s next to each other in the DNA sequence are stacked on top of each other in the actual DNA molecule. UV light can link them together to form a thymine dimer. This is a common effect of sunlight. • Thymine dimers distort the DNA double helix, and cause DNA polymerase to stall, so replication is blocked. • If not repaired, thymine dimers can cause various forms of skin cancer. • The genetic disease xeroderma pigmentosum is caused by the failure to repair thymine dimers. People with this disease develop freckles very easily, some of which turn cancerous. To avoid this, they need to stay out of direct sunlight. Repair Mechanisms • There are several different systems in the cell to repair different forms of damage to one strand of the DNA. However, most of them work in the same basic fashion: 1. Locate the damaged region 2. Excise the damaged and the surrounding nucleotides. This creates a free 3’ OH group that DNA polymerase can build off of. 3. Use a special DNA polymerase to resynthesize the damaged strand 4. Seal the nick remaining after DNA polymerase with DNA ligase. Mismatch Repair • DNA polymerase with its proofreading mechanism still makes about 1 error per 107 bp. (The human genome is about 3 x 109 bp). • The mismatch repair system fixes about 99% of these errors, giving an error rate of 1 in 109 bp. • It works like other DNA repair mechanisms. The initial step, recognizing the mismatch, occurs because mismatched bases form a slight bulge in the DNA double helix. • The tricky part is recognizing which strand still has the correct base and which has the incorrect base. • The incorrect base is on the newly synthesized strand • Current thought is that newly synthesized DNA has nicks in it, both in the lagging strand (between Okazaki fragments) and in the leading strand (presumably because DNA polymerase falls off the DNA and needs to be restarted). • The mismatch repair system recognizes which strand has nicks, and repairs it with the information from the other strand. Base Excision Repair • Many forms of DNA damage occur repeatedly; a good example is deamination of thymine to form uracil. These sites are easily dealt with by the base excision mechanism. • The cell contains several DNA glycosylases, which detect specific kinds of altered bases and cleaves the bond between the base and deoxyribose. This creates an abasic site. • Next, AP endonuclease cuts out the abasic nucleotide. This creates a short gap with a free 3’ –OH. • A special DNA polymerase resynthesizes the DNA in the region, and then DNA ligase seals the final nick. Nucleotide Excision Repair • Nucleotide excision repair works on DNA damage that none of the base excision repair glycosylases recognize: bulky alkyl groups attached to a base, thymine dimers, etc. • This system recognizes warps and bulges in the DNA double helix, then excises 1020 bases on either side of the problem, then uses DNA polymerase to resynthesize the second strand, and finally seals the remaining nick with DNA ligase. Double Stranded Breaks • If both strands of DNA break, there is no easy way to repair it: the ends just float freely away from each other. • One important mechanism of repairing double stranded breaks is non-homologous end joining. • Proteins bind to the broken DNA ends, then clean them by filling in single stranded gaps and making sure each 3’ end has a free –OH and each 5’ end has a free PO4 on it. The ends are then ligated together. • One of the proteins that binds to the broken chromosome ends in BRCA1. Mutations in this gene are a major cause of breast cancer. • If there are more than one pair of broken ends in the cell, this mechanism can easily join the wrong ones together. This is a major cause of chromosomal rearrangements like inversions and translocations. Repair by Homologous Recombination • A second way to repair double stranded breaks is to use the same mechanism used to perform crossing over in meiosis: homologous recombination. We will discuss crossing over later, but right now, we will examine how this process can repair a break. • The key is, in a diploid there are 2 copies of every sequence, one on each of the homologous chromosomes. If one chromosome is broken, you can use the information on the other one to repair it. • The first step is exonuclease activity that trims back the 5’ ends on both sides of the break, creating long single stranded ends with free 3’ -OH groups. • Next, the key step, strand invasion: the broken end of one DNA strand invades the unbroken double helix of the homologous chromosome and base pairs with it. • This broken end acts as a primer and a DNA polymerase extends it. • After some DNA synthesis, the elongated broken end is long enough to invade the other broken piece of DNA, and more DNA polymerase activity resynthesizes all the bases in the broken region. • DNA ligase then seals up the nicks. Steps in DNA Repair by Homologous Recombination, 1 1. Double stranded break in one homologue. Note that the bottom (blue) DNA molecule is written with the 3’->5’ strand on top, which is opposite of the usual way of writing it. 2. Cutting back of 5’ ends by an exonuclease, producing long tails with free 3’ ends. 3. Strand invasion: one 3’ end invades the double helix of the homologous DNA. 4. The strand that is displaced forms a Dloop (displacement loop) Steps in DNA Repair by Homologous Recombination, 2 5. The invading 3’ end is elongated by DNA polymerase, using the homologue as a template. The D-loop expands. 6. Capture of the other 3’ end by unpaired portion of the D-loop. 7. The captured 3’ end is elongated by DNA polymerase. 8. Ligation of free ends, creating 2 Holliday junctions (places where 2 DNA molecules joined together). 9. Resolution of the Holliday junctions by horizontal cuts and religation 10. Note that some sections of both DNA molecules are heteroduplex and thus possibly having some mispaired bases that will need to be repaired or resolved by replication. 11. Note that part of the broken DNA molecule has been converted to the sequence from the unbroken DNA molecule, with that area of the broken DNA lost. Homologous Recombination • Homologous recombination is the process by which 2 nearly identical DNA molecules break and rejoin to form new hybrid molecules. It is also called crossing over. • Homologous recombination is found in almost all organisms, in all 3 domains of life. It may have evolved from double strand break repair. • Homologous recombination creates new combinations of genes, which increases the chances of having a combination that is more evolutionarily fit than either parent. • In eukaryotes it occurs during meiosis (prophase of meiosis 1, for those who have taken my BIOS 308 class). In prokaryotes it can occur at any time. • The mechanism is the same as the homologous recombination repair mechanism, except for the very end. Homologous Recombination Mechanism,1 • Crossing over starts with a double stranded break, catalyzed by an enzyme (instead of a random event as in DNA repair). • The initial steps of recombination are identical to repair by homologous recombination. 1. Double stranded break in one homologue. 2. Cutting back of 5’ ends by an exonuclease, producing long tails with free 3’ ends. 3. Strand invasion: one 3’ end invades the double helix of the homologous DNA. 4. The strand that is displaced forms a Dloop. 5. The invading 3’ end is elongated by DNA polymerase. The D-loop expands. 6. Capture of the other 3’ end by unpaired portion of the D-loop. 7. The captured 3’ end is elongated by DNA polymerase. Homologous Recombination Mechanism,2 • Outside genetic markers A and B (on the red DNA) and a and b (on the blue DNA) have been added to make the results clearer. • However, there is a critical difference at resolution of the Holliday junctions. Rather than cutting both junctions horizontally, at the place where the strands cross, one junction is cut vertically (9A) 8. Ligation of the newly synthesized strands to the old strands. 9. Horizontal cut at one Holliday junction. 9A. Vertical cut at the other Holliday junction. 10A. The cut ends are ligated. The strands cross in this 2-dimensional drawing, so that the proper joins are made between 5’ and 3’ cut ends. 11A. After the DNA molecules are separated, it is clear that recombination has occurred: A on the red DNA (chromosome) is attached to b from the blue chromosome, and a from the blue is attached to B from the red. Holliday Junctions and the Results of Homologous Recombination • Two Holliday junctions appear during homologous recombination. They need to be resolved by cutting 2 strands and then ligating the cut ends so that the two DNA molecules can separate from each other. • The recombination of genetic markers outside the recombination site only occurs if one cut is horizontal and the other is vertical (as shown in the previous slide). • If both cuts are horizontal, or both are vertical, the outside markers (A, B, a, b) are not recombined: they stay linked together. Only a short region at the site of the recombination is heteroduplex. • If this happens within a gene, that gene can undergo gene conversion: it can switch from one allele (say, the red allele) to the other (the blue allele) without any recombination of markers outside the recombination site. • Gene conversion is rare, because most recombination occurs in DNA regions not part of any gene. In this picture of a Holliday junction and its resolution, what happens to the crossed strands is made easier to see by rotating the lower part of the junction.