28.7 How Is the Genetic Information Shuffled by Genetic Recombination? 883 (or single-strand uptake). Strand assimilation does not occur if there is no sequence homology between the ssDNA and the invaded DNA duplex. The DNA strand dis- placed by the invading 3Ј-terminal ssDNA is free to anneal with the 5Ј-terminal strand in the original DNA, a step that is also mediated by RecA protein and SSB (Figure 28.21c). The result is a Holliday junction, the classic intermediate in genetic recombination. RuvA, RuvB, and RuvC Proteins Resolve the Holliday Junction to Form the Recombination Products The Holliday junction is processed into recombination products by RuvA (203 amino acids), RuvB (336 amino acids), and RuvC (173 amino acids). Specifically, RuvA and RuvB work together as a Holliday junction–specific helicase complex that dissociates the RecA filament and catalyzes branch migration. An RuvA tetramer (Figure 28.22a) fits precisely within the junction point (Figure 28.22b), which has a square-planar geometry, and this RuvA tetramer mediates the assembly of RuvB around opposite arms of the DNA junction. The RuvB protein binds to form two oppositely oriented, hexameric [(RuvB) 6 ] ring structures encircling the dsDNAs, one on each side of the Holliday junction. Rotation of the dsDNAs by the RuvB hexameric rings pulls the dsDNAs through (RuvB) 6 and unwinds the DNA strands across the “spool” of RuvA, which threads the separated single strands into newly forming hybrid (recombinant) duplexes (Figure 28.22b). The RuvA tetramer is a disclike structure, one face of which has an overall positive charge (Figure 28.22c), with the exception of four negatively charged central pins, each contributed by a pair of residues (Glu 55 and Asp 56 ) from each RuvA monomer. These four pins fit neatly into the hole at the center of the Holliday junction. The negatively charged sugar–phosphate backbones of the four DNA duplexes of the Holliday junction are threaded along grooves in the positively charged RuvA face, with the negatively charged central pins appropriately situated to transiently separate the dsDNA molecules into their component single strands Junction binding Branch migration Resolution (b) RuvA RuvAB (a) RuvCRuvB (c) (d) ACTIVE FIGURE 28.22 Model for the resolution of a Holliday junction in E. coli by the RuvA, RuvB, and RuvC proteins. (a) Ribbon diagram of the RuvA tetramer. RuvA monomers have an overall L shape (pdb id ϭ 1CUK).(b) Model for RuvA/RuvB action (left).The RuvA tetramer fits snugly within the Holliday junction point. (center) Oppositely facing RuvB hexameric rings assemble on the heteroduplexes, with the DNA passing through their centers.These RuvB hexamers act as motors to promote branch migration by driving the passage of the DNA duplexes through themselves.(right) Binding of RuvC at the Holliday junction and strand scission by its nuclease activity.The locations of the RuvC active sites are indicated by the scissors.(c) Charge distribution on the concave surface of an RuvA tetramer.Blue indicates positive charge and red, negative charge.Note the overall positive charge on this surface of (RuvA) 4 , with the exception of the four red (negatively charged) pins at its center. (d) Structural model for the interaction of (RuvA) 4 with the hypothesized square-planar Holliday junction center (pdb id ϭ 1C7Y). (Adapted from Figures 1,2, and 3 in Rafferty, J. B., et al., 1996. Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction. Science 274:415–421.) Test yourself on the concepts in this figure at www.cengage.com/login. 884 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair through repulsive electrostatic interactions with the phosphate backbones of the DNA. The separated strands of each parental duplex are then channeled into grooves in the RuvA face, where they are led into hydrogen-bonding interactions with bases con- tributed by strands of the other parental DNA to form the two daughter hybrid du- plexes flowing out from the RuvAB complex (Figure 28.22b). Figure 28.22d illustrates a model for the RuvA tetramer with the square-planar Holliday junction. Depending on how the strands in the Holliday junction are cleaved and resolved, patch or splice recombinant duplexes result (Figure 28.18g and h). RuvC is an endo- A DEEPER LOOK The Three R’s of Genomic Manipulation: Replication, Recombination, and Repair DNA replication, recombination, and repair have traditionally been treated as separate aspects of DNA metabolism. However, scientists have come to realize that DNA replication is an essen- tial component of both DNA recombination and DNA repair processes. Furthermore, recombination mechanisms play an ab- solutely vital role in restarting replication forks that become halted at breaks or other lesions in the DNA strands. If a double- stranded break (DSB) or a nick in just one of the DNA strands (called a single-stranded gap, or SSG) is present in the DNA under- going replication, the replication fork stalls and the replication complex dissociates (replication fork “collapse”). Significantly, the whole process of homologous recombination can initiate only at SSGs or DSBs, and establishment of homologous recombination at such sites can rescue DNA replication. This recombination- dependent replication (RDR) has the interesting property of ini- tiating DNA replication at sites other than the oriC site, and thus RDR is an important mechanism for restarting DNA replication if the replication fork is disrupted for any reason. As might be ex- pected from the close relationships between replication, recom- bination, and repair, many of the same proteins are involved in all three, and all three must be viewed as essential processes in the perpetuation of the genome. A DEEPER LOOK “Knockout” Mice: A Method to Investigate the Essentiality of a Gene Homologous recombination can be used to replace a gene with an inactivated equivalent of itself. Inactivation is accomplished by in- serting a foreign gene, such as neo, a gene encoding resistance to the drug G418, within one of the exons of a copy of the gene of in- terest. Homologous recombination between the neo-bearing trans- gene and DNA in wild-type mouse embryonic stem (ES) cells replaces the target gene with the inactive transgene (see accom- panying figure). ES cells in which homologous recombination has occurred will be resistant to G418, and such cells can be selected. These recombinant ES cells can then be injected into early-stage mouse embryos, where they have a chance of becoming the germline cells of the newborn mouse. If they do, an inactivated target gene is then present in the gametes of this mouse. Mating between male and female mice with inactive target genes yields a generation of homozygous “knockout” mice—mice lacking a func- tional copy of the targeted gene. Characterization of these knock- out mice reveals which physiological functions the gene directs. 1 1 2 3 3 Exons Copy of wild-type gene trans gene neo 1 3 neo 13 13 neo Insertion of neo into exon 2 Transfection into ES cell Homologous recombination Host target gene Host target gene disrupted by neo-bearing trans gene 2 28.7 How Is the Genetic Information Shuffled by Genetic Recombination? 885 nuclease that resolves Holliday junctions into heteroduplex recombinant products (RuvC resolvase). An RuvC dimer binds at the Holliday junction and cuts pairs of DNA strands of similar polarity (Figure 28.22b); whether a patch or a splice recom- binant results depends on which DNA pair is cleaved. RuvB hexamers are AAA ϩ -ATPase-type, DNA-driving molecular motors; similar motors act during DNA replication to propel strand separation and initiate DNA synthesis. Thus, the RuvABC system for processing Holliday junctions may repre- sent a general paradigm for DNA manipulation in all cells. Recombination-Dependent Replication Restarts DNA Replication at Stalled Replication Forks It is likely that most replication forks that begin at the E. coli oriC initiation sites (or analogous initiation sites in eukaryotes) are derailed by nicks or more extensive DNA damage lying downstream. However, DNA replication can be reinitiated (and genome replication can be completed) following replication fork restart. Recombi- national repair of stalled replication forks requires the action of enzymes from every aspect of DNA metabolism: replication, recombination, and repair. The initial steps in restoration of a replication fork depend on the recombination proteins RecA and RecBCD and the formation of a D-loop (Figure 28.21). The E. coli protein PriA rec- ognizes and binds with high affinity to D-loops. Once bound, PriA coordinates resumption of DNA replication by recruiting DnaB helicase to the D-loop and reestablishing a replication fork complete with two copies of the replicative DNA polymerase. The ability of RecA to mediate recombinational repair of stalled repli- cation forks is undoubtedly the reason for the presence of RecA-type proteins in vir- tually all organisms. Indeed, the recombination system, a feature common to all cells, evolved to carry out this essential repair purpose. Transposons Are DNA Sequences That Can Move from Place to Place in the Genome In 1950, Barbara McClintock reported the results of her studies on an activator gene in maize (Zea mays, or as it’s usually called, corn) that was recognizable principally by its ability to cause mutations in a second gene. Activator genes were thus an internal source of mutation. A most puzzling property was their ability to move relatively freely HUMAN BIOCHEMISTRY The Breast Cancer Susceptibility Genes BRCA1 and BRCA2 Are Involved in DNA Damage C ontrol and DNA Repair Mutations in the BRCA1 and BRCA2 genes cause increased likeli- hood of breast, ovarian, and other cancers. The BRCA1 protein functions in regulation of the cell cycle in response to DNA damage control. Phosphorylation of BRCA1 by DNA damage-response pro- teins controls the expression, phosphorylation, and cellular local- ization of specific cyclin-CDKs involved in the cell cycle G 2 /M checkpoint and the onset of mitosis. Activation of these cyclin-CDKs leads to arrest of the cell cycle in G 2 until the damage to DNA is re- paired. Mutations in BRCA1 that impair its function allow the cell cycle to enter mitosis and DNA damage to accumulate, raising the risk of cancer. The BRCA2 protein participates in the pathway for DNA repair by homologous recombination. The BRCA2 protein (3418 amino acids) is a very large protein with 8 conserved sequence motifs of about 30 amino acids each, known as the BRC repeats. These re- peats act as binding sites for RAD51, the eukaryotic analog of RecA. BRCA2 transfers RAD51 to a ssDNA strand coated with RPA (the eukaryotic counterpart of SSB), allowing formation of the RAD51–ssDNA nucleoprotein filament that is an essential inter- mediate in eukaryotic homologous recombination (see Figure 28.20b). Mutations that impair BRCA2 function prevent DNA re- pair by homologous recombination, leading to accumulation of DNA damage and a greater likelihood of cancer. Source: Yarden, R. I., et al., 2003. BRCA1 regulates the G 2 /M checkpoint by activating Chk1 kinase upon DNA damage. Nature Genetics 30:285–289; Simon, N., Powell, S. M., Willers, H., and Xia, F., 2003. BRCA2 keeps Rad51 in line: High-fidelity homologous recombination prevents breast and ovar- ian cancer? Molecular Cell 10:1262–1263; and Pelligrini, L., et al., 2002. In- sights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature 420:287–293. 886 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair about the genome. Scientists had labored to establish that chromosomes consisted of genes arrayed in a fixed order, so most geneticists viewed as incredible this idea of genes moving around. The recognition that McClintock so richly deserved for her explanation of this novel phenomenon had to await verification by molecular biolo- gists. In 1983, Barbara McClintock was finally awarded the Nobel Prize in Physiology or Medicine. By this time, it was appreciated that many organisms, from bacteria to hu- mans, possessed similar “jumping genes” able to move from one site to another in the genome. This mobility led to their designation as mobile elements, transposable elements, or, simply, transposons. Transposons are segments of DNA that are moved enzymatically from place to place in the genome (Figure 28.23). That is, their location within the DNA is unsta- ble. Transposons range in size from several hundred base pairs to more than 8 kbp. Transposons contain a gene encoding an enzyme necessary for insertion into a chro- mosome and for the remobilization of the transposon to different locations. These movements are termed transposition events. The smallest transposons are called insertion sequences, or ISs, signifying their ability to insert apparently at random in the genome. Insertion into a new site can cause a mutation if a gene or regulatory re- gion at the site is disrupted. Because transposition events can move genes to new places or lead to the duplication of existing genes, transposition is a major force in evolution. Transposon ACGTACGTACGT ACGTACGTACGT TGCATGCATGCA TGCATGCATGCA ACGTACGTACGT ACGTACGTACGT TGCATGCATGCA TGCATGCATGCA CATGC GTACG CATGC CATGC GTACG GTAGC ACGTACGTACGT ACGTACGTACGT TGCATGCATGCA CATGC GTACG Target site Host DNA Staggered cuts at target site TGCATGCATGCA CATGC GTACG (a) Gaps filled in and strands ligated Insertion of transposon Inverted repeats Target site direct repeats (b) (c) (d) ACTIVE FIGURE 28.23 The typical transposon has inverted nucleotide-sequence repeats at its termini, represented here as the 12-bp sequence ACGTACGTACGT (a). It acts at a target sequence (shown here as the sequence CATGC) within host DNA by creat- ing a staggered cut (b) whose protruding single- stranded ends are then ligated to the transposon (c). The gaps at the target site are then filled in, and the filled-in strands are ligated (d). Transposon insertion thus generates direct repeats of the target site in the host DNA, and these direct repeats flank the inserted transposon. Test yourself on the concepts in this figure at www.cengage.com/login. 28.8 Can DNA Be Repaired? 887 28.8 Can DNA Be Repaired? Biological macromolecules are susceptible to chemical alterations that arise from environmental damage or errors during synthesis. For RNAs, proteins, or other cel- lular molecules, most consequences of such damage are avoided by replacement of these molecules through normal turnover (synthesis and degradation). However, the integrity of DNA is vital to cell survival and reproduction. Its information con- tent must be protected over the life span of the cell and preserved from generation to generation. Safeguards include (1) high-fidelity replication systems and (2) re- pair systems that correct DNA damage that might alter its information content. DNA is the only molecule that, if damaged, is repaired by the cell. Usually, accurate repair is possible because the information content of duplex DNA is inherently re- dundant; the nucleotide sequence in one strand is directly related to the sequence in the other. DNA damage may arise from endogenous processes or from exoge- nous agents, such as UV light, ionizing radiation, or mutagenic chemicals. The most common forms of endogenous DNA damage arise from chemical reactions (oxi- dation, alkylation, or deamination of bases) or loss of bases due to cleavage of N-glycosidic bonds. Exogenous agents can damage DNA in a variety of ways, in- cluding UV-induced free-radical generation and crosslinking of adjacent pyrim- idines, breakage of the polynucleotide backbone by ionizing radiation, and base modifications through chemical reactions. Cells have extraordinarily diverse and ef- fective systems to repair these lesions in DNA so that the genetic information is not lost or altered. The human genome has 150 or so genes associated with DNA repair. DNA repair systems include direct reversal damage repair, single-strand damag e re- pair, double-stranded break (DSB) repair, and translesion DNA synthesis. Chemical reactions that reverse the damage, returning the DNA to its proper state, are called direct reversal repair systems. Examples include methyltransferases to remove methyl groups from chemically modified bases and photolyase, which repairs thymine dimers, as discussed in a later section (see Figure 28.27). Single- strand damage repair relies on the intact complementary strand to guide repair. Sys- tems repairing this sort of DNA damage include mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER). Such systems will be described further on. DSBs are a particular threat to genome stability, because lost sequence informa- tion cannot be recovered from the same DNA double helix. Because the threat is so severe, cells have several systems to repair DSBs. The simplest way to repair a DSB is to rejoin the broken strands through nonhomologous DNA end-joining, or NHEJ (Figure 28.24). This repair can occur any time in the cell cycle. A key problem in NHEJ is to keep the ends near one another so that the fragments can be linked to- gether again. A heterodimeric protein, Ku70/80, binds the DNA ends and recruits a set of proteins that juxtaposes the damaged ends, repairs them, and religates them without the use of a repair template. (Some proteins involved in repairing DSBs also function in immunoglobulin gene rearrangements; see Figure 28.39.) The lack of a proper template for NHEJ means that it is error-prone. DSBs that arise during the S phase of the cell cycle can be repaired through ho- mologous recombination (Figure 28.25). The intact sister chromatid of the dam- aged DNA duplex guides the process. Processing of the DSB creates single-stranded tails that become substrates for RecA-mediated nucleoprotein filament formation and homology recognition within the sister chromatid. DNA synthesis propagated from the D-loop and migration of the Holliday junction, followed by strand cleav- ag e within the Holliday junction by resolvase, either restores the DNA to its intact state (Figure 28.25g, left) or results in strand exchange between the two sister chro- matids (Figure 28.25g, right). What is effectively a DSB can arise during DNA replication if DNA damage causes the replication fork to stall (Figure 28.26). Suppose a lesion in the leading strand (circle, Figure 28.26b) causes leading-strand synthesis to stall. Continued lagging-strand synthesis results in a single-stranded region on the leading-strand DSB Ku 70/80 DNA-dependent protein kinase, end-processing enzymes, DNA ligase IV FIGURE 28.24 DSB repair through nonhomologous DNA end joining (NHEJ). Ku70/80 binds the ends and recruits a set of proteins that juxtaposes the broken ends. Pro- cessing of the ends to generate proper substrates for DNA ligase IV then occurs, followed by DNA-ligase- mediated end joining. (Adapted from Figure 1 in Wyman, C., and Kanaar, R., 2006. DNA double-strand break repair: All’s well that ends well. Annual Review of Genetics 40:363–383.) 888 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair template. Leading-strand invasion of the new lagging-strand duplex (mediated by RecA) creates a D-loop (Figure 28.26c), and leading-strand synthesis directed by the lagging-strand template (Figure 29.26d) reestablishes a competent replication fork (Figure 28.26e). The site of the lesion is repaired later, usually by nucleotide excision repair (see following section). Homologous recombination-mediated restarting of replication forks is believed to be the evolutionary driving force for the emergence of homologous recombination. Even if the NHEJ or homologous recombination repair systems fail to act, the genome may be preserved if an “error- prone” mode of replication allows the lesion to be bypassed. Such translesion DNA (a) (b) (c) (d) (e) (f) (g) (a) (b) (c) (e) (d) FIGURE 28.25 DSB repair through homologous DNA recombination.The orange–red pair of lines symbolizes the double-stranded DNA with a DSB; the black–blue pair represents the sister chromatid. Homologous recombination creates a D-loop (c), and sister chromatid- directed DNA replication restores the information content of the damaged duplex (d–f). Depending on how the Holliday junctions are resolved, the products (g) are either (left) noncrossover or (right) crossover recombinants.(Adapted from Figure 2 in Wyman, C., and Kanaar, R., 2006. DNA double-strand break repair: All’s well that ends well. Annual Review of Genetics 40:363–383.) FIGURE 28.26 Restarting a stalled replication fork through ho- mologous DNA recombination. A lesion in the DNA is symbol- ized by a circle; in this case, the lesion is in the leading-strand template (a). Leading-strand synthesis halts because of the le- sion (b). Lagging-strand synthesis (red) continues, and the Okazaki fragments are ligated (c). When the leading strand in- vades the new DNA duplex formed by lagging-strand synthesis, a D-loop is formed and strand exchange occurs. Using the lag- ging strand as a template, synthesis of the leading strand (black) resumes (d), and the replication fork is reestablished (e). (Adapted from Figure 3 in Wyman, C., and Kanaar, R., 2006. DNA double-strand break repair: All’s well that ends well.Annual Review of Genetics 40:363–383.) 28.8 Can DNA Be Repaired? 889 synthesis is more of a tolerance mechanism than a repair mechanism because it al- lows replication without necessarily repairing the damage. Specialized translesion DNA polymerases, such as DNA polymerase IV in E. coli and DNA polymerase ( ϭ eta) in humans, substitute for the replicative DNA polymerase in this process. Although translesion DNA polymerases tend to be error-prone, they have the ad- vantage of allowing DNA replication to continue. Human DNA replication has an error rate of about three base-pair mistakes dur- ing copying of the 6 billion base pairs in the diploid human genome. The low error rate is due to those DNA repair systems that review and edit the newly replicated DNA. Furthermore, about 10 4 bases (mostly purines) are lost per cell per day from A DEEPER LOOK Transgenic Animals Are Animals Carrying Foreign Genes Experimental advances in gene transfer techniques have made it possible to introduce genes into animals by transfection. Transfec- tion is defined as the uptake or injection of plasmid DNA into re- cipient cells. Animals that have acquired new genetic information as a consequence of the introduction of foreign genes are termed transgenic. Plasmids carrying the gene of interest are injected into the nucleus of an oocyte or fertilized egg, and the egg is then im- planted into a receptive female. The technique has been perfected for mice (see figure, part a). In a small number of cases—10% or so—the mice that develop from the injected eggs carry the trans- fected gene integrated into a single chromosomal site. The gene is subsequently inherited by the progeny of the transfected animal as if it were a normal gene. Expression of the donor gene in the trans- genic animals is variable because the gene is randomly integrated into the host genome and gene expression is often influenced by chromosomal location. Nevertheless, transfection of animals has produced some startling results, as in the case of the transfection of mice with the gene encoding the rat growth hormone (rGH). The transgenic mice grew to nearly twice the normal size (see figure, part b). Growth hormone levels in these animals were several hun- dred times greater than normal. Similar results were obtained in transgenic mice transfected with the human growth hormone (hGH) gene. The biotechnology of transfection has been extended to farm animals, and transgenic chickens, cows, pigs, rabbits, sheep, and even fish have been produced. The first animal cloned from an adult cell, a sheep named Dolly, represented a milestone in cloning technolo gy. Subsequent accomplishments include incorporation of the human gene en- coding blood coagulation factor IX into sheep. Fetal sheep fi- broblast cells were transfected with the human factor IX gene, nu- clei from the transfected cells were transferred into sheep oocytes lacking nuclei, and these transgenic oocytes were placed in the uterus of receptive female sheep, which subsequently gave birth to transgenic lambs. The introduced factor IX transgene was specifi- cally designed so that factor IX protein, a medically useful product for the treatment of hemophiliacs, would be expressed in the milk of the transgenic sheep. Similar successes in cows, which produce much more milk, has brought the potential for commercial pro- duction of virtually any protein into the realm of reality. Transfection technology also holds promise as a mechanism for “gene therapy” by replacing defective genes in animals with func- tional genes (see Chapter 12). Problems concerning delivery, in- tegration and regulation of the transfected gene, including its ap- propriate expression in the right cells at the proper time during development and growth of the organism, must be brought under control before gene therapy becomes commonplace in humans. Plasmid carrying growth hormone gene from rat Mouse egg Injected into Egg implanted into mouse Mouse gives birth Extract DNA from tissue biopsy If rat growth hormone DNA present, assay for rat growth hormone in mouse tissue Hybridize with probe for plasmid by Southern blotting nucleus Mate transgenic mouse to obtain progeny (a) (b) ᮡ (a) Transfection can introduce new genes into animals. The rat growth hormone gene carried on a plasmid is injected into a mouse oocyte or fertilized egg that is then implanted in a receptive female mouse. Integration of the plasmid into the mouse genome can be ascertained by Southern blot analysis of DNA from the newborn mouse. Expression of the foreign gene can be determined by assaying for the gene product, in this case, rat growth hormone. (b) Photograph showing a transgenic mouse with an active rat growth hormone gene (left). This transgenic mouse is twice the size of a normal mouse (right). Courtesy of Ralph L. Brinster, School of Veterinary Medicine, University of Pennsylvania 890 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair spontaneous breakdown in human DNA; repair systems must replace these bases to maintain the fidelity of the encoded information. Mismatch Repair Corrects Errors Introduced During DNA Replication The mismatch repair system corrects errors introduced when DNA is replicated. It scans newly synthesized DNA for mispaired bases, excises the mismatched region, and then replaces it by DNA polymerase–mediated local replication. The key to such re- placement is to know which base of the mismatched pair is correct. The E. coli methyl-directed pathway of mismatch repair relies on methylation pat- terns in the DNA to determine which strand is the newly synthesized one and which one was the parental (template) strand. DNA methylation, often an identifying and characteristic feature of a prokaryote’s DNA, occurs just after DNA replication. Dur- ing methylation, methyl groups are added to certain bases along the new DNA strand. However, a window of opportunity exists between the start of methylation and the end of replication, when only the parental strand of a dsDNA is methylated. This window in time provides an opportunity for the mismatch repair system to review the dsDNA for mismatched bases that arose as a consequence of replication errors. By definition, the newly synthesized strand is the one containing the error, and the methylated strand is the one having the correct nucleotide sequence. When the methyl-directed mismatch repair system encounters a mismatched base pair, it searches along the DNA—through thousands of base pairs if necessary—until it finds a methylated base. The system identifies the strand bearing the methylated base as parental, as- sumes its sequence is the correct one, and replaces the entire stretch of nucleotides within the new strand from this recognition point to and including the mismatched base. Mismatch repair does this by using an endonuclease to cut the new, un- methylated strand and an exonuclease to remove the mismatched bases, creating a gap in the newly synthesized strand. DNA polymerase III holoenzyme then fills in the gap, using the methylated strand as template. Finally, DNA ligase reseals the strand. Damage to DNA by UV Light or Chemical Modification Can Also Be Repaired Repair of Pyrimidine Dimers Formed by UV Light UV irradiation promotes the for- mation of covalent bonds between adjacent thymine residues in a DNA strand, creat- ing a cyclobutyl ring (Figure 28.27). Because the COC bonds in this ring are shorter than the normal 0.34-nm base stacking in B-DNA, the DNA is distorted at this spot and is no longer a proper template for either replication or transcription. Photolyase (also called photoreactivating enzyme), a flavin- and pterin-dependent enzyme, binds at the dimer and uses the energy of visible light to break the cyclobutyl ring, restor- ing the pyrimidines to their original form. CN CN CC O H O HCH 3 CN CN CC O H O HCH 3 C N CN C C O H O CH 3 CN CN H C C O H O CH 3 H Sugar Sugar Phosphate UV Cyclobutyl ring Sugar Sugar Phosphate FIGURE 28.27 UV irradiation causes dimerization of adja- cent thymine bases. A cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers. 28.9 What Is the Molecular Basis of Mutation? 891 Excision Repair Replacement of chemically damaged or modified bases occurs via two fundamental excision repair systems—base excision and nucleotide excision. Base excision repair acts on single bases that have been damaged through oxidation or other chemical modifications during normal cellular processes. The damaged base is removed by DNA glycosylase, which cleaves the glycosidic bond, creating an apurinic acid (AP) site where the sugar–phosphate backbone is intact but a purine (apurinic site) or a pyrimidine (apyrimidinic site) is missing. An AP endonuclease then cleaves the backbone, an exonuclease removes the deoxyribose-P and a number of additional residues, and the gap is repaired by DNA polymerase and DNA ligase (Figure 28.28). The information of the complementary strand is used to dictate which bases are added in refilling the gap. In E. coli, DNA polymerase I binds at the gap and moves in the 5Ј→3Ј direction, removing nucleotides with its 5Ј-exonuclease activity. The 5Ј→3Ј DNA polymerase activity of DNA polymerase I fills in the sequence behind the 5Ј-exonuclease action. No net synthesis of DNA results, but this action of DNA po- lymerase I “edits out” sections of damaged DNA. Excision is coordinated with 5Ј→3Ј polymerase-catalyzed replacement of the damaged nucleotides so that DNA of the right sequence is restored. Nucleotide excision repair recognizes and repairs larger regions of damaged DNA than base excision repair. The nucleotide excision repair system cuts the sugar– phosphate backbone of a DNA strand in two places, one on each side of the lesion, and removes the region. The region removed in prokaryotic nucleotide excision re- pair spans 12 or 13 nucleotides; in eukaryotic excision repair, an oligonucleotide stretch 27 to 29 units long is removed. The resultant gap is then filled in using DNA polymerase (DNA polymerase I in prokaryotes or DNA polymerase ␦ or ⑀ and PCNA plus RFC in eukaryotes), and the sugar–phosphate backbone is covalently closed by DNA ligase. In mammalian cells, nucleotide excision repair is the main pathway for removal of carcinogenic (cancer-causing) lesions caused by sunlight or other mutagenic agents. Such lesions are recognized by XPA protein, named for xeroderma pigmentosum, an in- herited human syndrome whose victims suffer serious skin lesions if exposed to sun- light. At sites recognized by XPA, a multiprotein endonuclease is assembled and the damaged strand is cleaved and repaired. 28.9 What Is the Molecular Basis of Mutation? Genes are normally transmitted unchanged from generation to generation, owing to the great precision and fidelity with which genes are copied during chromosome du- plication. However, on rare occasions, genetically heritable changes (mutations) occur and result in altered forms. Most mutated genes function less effectively than the un- altered, wild-type allele, but occasionally mutations arise that give the organism a se- lective advantage. When this occurs, they may be propagated to many offspring. To- gether with recombination, mutation provides for genetic variability within species and, ultimately, the evolution of new species. Mutations change the sequence of bases in DNA, either by the substitution of one base pair for another (so-called point mutations) or by the insertion or deletion of one or more base pairs (insertions and deletions). Point Mutations Arise by Inappropriate Base-Pairing Point mutations arise when a base pairs with an inappropriate partner. The two pos- sible kinds of point mutations are transitions, in which one purine (or pyrimidine) is replaced by another, as in A⎯→G (or T⎯→C), and transversions, in which a purine is substituted for a pyrimidine, or vice versa. Point mutations arise by the pairing of bases with inappropriate partners dur- ing DNA replication, by the introduction of base analogs into DNA, or by chemi- cal mutagens. Bases may rarely mispair (Figure 28.29), either because of their tautomeric properties (see Chapter 10) or because of other influences. Even in C GA T A C G T C GA T A C G T C GA T A C G T T T A C G A TC G C G T Damaged base DNA glycosylase T A C G A TC G C G Apurinic/ apyrimidinic endonuclease T A C G A TC G C G T Excision exonuclease Polymerase AP site C GA T A C G T T A C G A T A C G C G T C GA T A C G T T A C G A TC G New DNA Ligase C GA T A C G T T A C G A T A C G C G T FIGURE 28.28 Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site.Then, an apurinic/ apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap. 892 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair mispairing, the C 1 Ј–C 1 Ј distances between bases must still be close to that of a Watson–Crick base pair (11 nm or so; see Figure 11.6) to maintain the mis- matched base pair in the double helix. In tautomerization, for example, an amino group (ONH 2 ), usually an H-bond donor, can tautomerize to an imino form (PNH) and become an H-bond acceptor. Or a keto group (CPO), normally an H-bond acceptor, can tautomerize to an enol COOH, an H-bond donor. Proof- reading mechanisms operating during DNA replication catch most mispairings. The frequency of spontaneous mutation in both E. coli and fruit flies (Drosophila melanogaster) is about 10 Ϫ10 per base pair per replication. Mutations Can Be Induced by Base Analogs Base analogs that become incorporated into DNA can induce mutations through changes in base-pairing possibilities. Two examples are 5-bromouracil (5-BU) and 2-aminopurine (2-AP). 5-Bromouracil is a thymine analog and becomes inserted into DNA at sites normally occupied by T; its 5-Br group sterically resembles thymine’s 5-methyl group. However, because 5-BU frequently assumes the enol tautomeric form and pairs with G instead of A, a point mutation of the transition type may be induced T (1) CH 3 O O N H N N HH N N N CЈ 1 CЈ 1 CЈ 1 CЈ 1 CЈ 1 CЈ 1 Cytosine (2) H O H N H N N H H N N N CЈ 1 CЈ 1 Rare imino tautomer of adenine N H 1 6 N CH 3 O O O O N H H H H H H H H N TA TA CA * TA TA TA CG (3) N N N N N N N N H N N N N N A G (c) (b) (a) A TC N FIGURE 28.29 Point mutations due to base mispairings.(a) An example based on tautomeric properties.The rare imino tautomer of adenine base pairs with cytosine rather than thymine. (1) The normal A-T base pair. (2) The A*–C base pair is possible for the adenine tautomer in which a proton has been transferred from the 6-NH 2 of adenine to N-1. (3) Pairing of C with the imino tautomer of A (A*) leads to a transition mutation (A–T to G–C) appearing in the next generation. (b) A in the syn conformation pairing with G (G is in the usual anti con- formation). (c) T and C form a base pair by H-bonding interactions mediated by a water molecule. O Br H N N O O Br H N N OH H H N O N N N N 5-Bromouracil (5-BU) (keto tautomer) 5-BU (enol tautomer) Guanine CЈ 1 CЈ 1 CЈ 1 FIGURE 28.30 5-Bromouracil usually favors the keto tautomer that mimics the base-pairing properties of thymine, but it frequently shifts to the enol form, where- upon it can base-pair with guanine, causing a T–A to C–G transition.