Bio3500CH.12 DNA Replication PDF

## 12.1 Genetic Information Must Be Accurately Copied Every Time a Cell Divides In a schoolyard game, a verbal message like "John's brown dog ran away from home," is whispered to a child. This child runs to another child and repeats the message. The message is relayed from child to child until it r...

## 12.1 Genetic Information Must Be Accurately Copied Every Time a Cell Divides In a schoolyard game, a verbal message like "John's brown dog ran away from home," is whispered to a child. This child runs to another child and repeats the message. The message is relayed from child to child until it returns to the original sender. Eventually, the last child returns with a vastly changed message, such as "Joe Brown has a pig living under his porch." The more children playing the game, the more garbled the message becomes. This game illustrates an important principle: errors arise whenever information is copied, and the more times it is copied, the greater the potential number of errors. A complex multicellular organism faces a problem analogous to that of the children in the schoolyard game: how to faithfully transmit genetic instructions each time its cells divide. The solution to this problem is central to replication. A single-celled human zygote contains 6.4 billion base pairs of DNA; even a low rate of error during copying, such as once per million base pairs, would result in 6400 mistakes made every time a cell divided - errors that would be compounded at each of the millions of cell divisions that take place in human development. Not only must the copying of DNA be astoundingly accurate, it must also take place at rapid speed. The single circular chromosome of *Escherichia coli* contains about 4.6 million base pairs. At a rate of 1000 nucleotides per minute, replication of the entire chromosome would require over 3 days. Yet these bacteria are capable of dividing every 20 minutes. *E. coli* actually replicates its DNA at a rate of 1000 nucleotides per second, with less than one error in a billion nucleotides. How is this extraordinarily accurate and rapid process accomplished? ## 12.2 All DNA Replication Takes Place in a Semiconservative Manner When Watson and Crick solved the three-dimensional structure of DNA in 1953, several important genetic implications were immediately apparent. The complementary nature of the two nucleotide strands in a DNA molecule suggested that during replication, each strand can serve as a template for the synthesis of a new strand. The specificity of base pairing (adenine with thymine, guanine with cytosine) implied that only one sequence of bases can be specified by each template strand, and so the two DNA molecules built on the pair of templates will be identical to the original. This process is called **semiconservative replication** because each of the original nucleotide strands remains intact (conserved), despite their no longer being combined in the same molecule; thus, the original DNA molecule is half (semi-) conserved during replication. Initially, three models were proposed for DNA replication. - **Conservative replication**: The entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA, and the original DNA molecule is fully conserved during replication. - **Dispersive replication**: Both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules. In this model, each resulting DNA molecule contains interspersed fragments of old and new DNA; none of the original molecule is conserved. - **Semiconservative replication**: The two nucleotide strands unwind, and each serves as a template for a new DNA molecule. These three models allow different predictions to be made about the distribution of original DNA and newly synthesized DNA after replication. - **Conservative replication**: After one round of replication, 50% of the molecules would consist entirely of the original DNA and 50% would consist entirely of new DNA. After a second round of replication, 25% of the molecules would consist entirely of the original DNA and 75% would consist entirely of new DNA. With each additional round of replication, the proportion of molecules with new DNA would increase, although the number of molecules with the original DNA would remain constant. - **Dispersive replication**: Would always produce hybrid molecules, containing some original and some new DNA, but the proportion of new DNA within the molecules would increase with each replication event. - **Semiconservative replication**: One round of replication would produce two hybrid molecules, each consisting of half original DNA and half new DNA. After a second round of replication, half the molecules would be hybrid and the other half would consist of new DNA only. Additional rounds of replication would produce more and more molecules consisting entirely of new DNA, but a few hybrid molecules would persist. ### Meselson and Stahl's Experiment To determine which of the three models of replication applied to *E. coli* cells, Matthew Meselson and Franklin Stahl needed a way to distinguish old and new DNA. They accomplished this by using two isotopes of nitrogen, ^(14)N (the common form) and ^(15)N (a rare, heavy form). Meselson and Stahl grew a culture of *E. coli* in a medium that contained ^(15)N as the sole nitrogen source; after many generations, all the *E. coli* cells contained ^(15)N incorporated into all the purine and pyrimidine bases of their DNA. They took a sample of these bacteria, switched the rest of the bacteria to a medium that contained only ^(14)N, and then took additional samples of bacteria over the next few cellular generations. In each sample, the bacterial DNA that was synthesized before the change in medium contained ^(15)N and was relatively heavy, whereas any DNA synthesized after the switch contained ^(14)N and was relatively light. Meselson and Stahl distinguished between the heavy ^(15)N-laden DNA and the light ^(14)N-containing DNA with the use of **equilibrium density gradient centrifugation**. In this technique, a centrifuge tube is filled with a heavy salt solution and a substance of unknown density - in this case, DNA fragments. The tube is then spun in a centrifuge at high speeds. After several days of spinning, a gradient of density develops within the tube, with high-density material at the bottom and low-density material at the top. The density of the DNA fragments matches that of the salt: light molecules rise and heavy molecules sink. Meselson and Stahl found that DNA from bacteria grown only in a medium containing ^(15)N produced a single band at the position expected of DNA containing only ^(15)N. DNA from bacteria transferred to the medium with ^(14)N and allowed one round of replication also produced a single band, but at a position intermediate between that expected of DNA containing only ^(15)N and that expected of DNA containing only ^(14)N. This result is inconsistent with the conservative replication model, which predicts one heavy band (the original DNA molecules) and one light band (the new DNA molecules). A single band of intermediate density is predicted by both the semiconservative and the dispersive models. To distinguish between these two models, Meselson and Stahl grew the bacteria in a medium containing ^(14)N for a second generation. After a second round of replication in a medium with ^(14)N, two bands of equal intensity appeared, one in the intermediate position and the other at the position expected of DNA containing only ^(14)N. All samples taken after additional rounds of replication produced the same two bands, and the band representing light DNA became progressively stronger. Meselson and Stahl's results were exactly as expected for semiconservative replication and were incompatible with those predicted for both conservative and dispersive replication. ## Modes of Replication After Meselson and Stahl's work was published, investigators confirmed that other organisms also use semiconservative replication. No evidence was found for conservative or dispersive replication. There are, however, several different ways in which semiconservative replication can take place, differing principally in the nature of the template DNA - that is, whether it is linear or circular. A segment of DNA that undergoes replication is called a **replicon**, and each replicon contains an **origin of replication**. Replication starts at the origin and continues until the entire replicon has been replicated. Bacterial chromosomes have a single origin of replication, whereas eukaryotic chromosomes contain many. ### Theta Replication A common mode of replication that takes place in circular DNA, such as that found in *E. coli* and other bacteria, is called **theta replication** because it generates an intermediate structure that resembles the Greek letter theta (θ). In Figure 12.4a and all subsequent figures in this chapter, the original (template) strand of DNA is shown in gray and the newly synthesized strand of DNA is shown in red. In theta replication, double-stranded DNA begins to unwind at the origin of replication, producing single nucleotide strands that then serve as templates on which new DNA can be synthesized. The unwinding of the double helix generates a loop, termed a **replication bubble**. Unwinding may occur at one or both ends of the bubble, making it progressively larger. DNA replication on both of the template strands is simultaneous with unwinding. The point of unwinding, where the two strands separate from the double-stranded DNA helix, is called a **replication fork**. If there are two replication forks, one at each end of the replication bubble, the forks proceed outward in both directions in a process called **bidirectional replication**, simultaneously unwinding and replicating the DNA until they eventually meet. If unidirectional replication with a single replication fork is present, it proceeds around the entire circle. Both bidirectional and unidirectional replication produce two complete circular DNA molecules, each consisting of one old and one new nucleotide strand. John Cairns provided the first visible evidence of theta replication in 1963 by growing bacteria in the presence of radioactive nucleotides. After replication, each DNA molecule consisted of one "hot" (radioactive) strand and one "cold" (nonradioactive) strand. Cairns isolated DNA from the bacteria after replication, placed it on an electron-microscope grid, and then covered it with a photographic emulsion. Radioactivity present in the sample exposed the emulsion and produced a picture of the molecule (called an autoradiograph) in a manner similar to the light that exposes a photographic film. Because the newly synthesized DNA contained radioactive nucleotides, Cairns was able to produce electron micrographs of the replication process similar to those shown in Figure 12.4b. ### Rolling-Circle Replication Another form of replication, called **rolling-circle replication** takes place in some viruses and in the F factor of *E. coli* (a small circle of extrachromosomal DNA that controls mating, discussed in Section 9.3). This form of replication is initiated by a break in one of the nucleotide strands, which exposes a 3'-OH group and a 5'-phosphate group. New nucleotides are added to the 3' end of the broken strand, using the inner (unbroken) strand as a template. As new nucleotides are added to the 3' end, the 5' end of the broken strand is displaced from the template, rolling out like thread being pulled off a spool. The 3' end grows around the circle, giving rise to the name rolling-circle replication. Circular DNA molecules that undergo theta or rolling-circle replication have a single origin of replication. Because of the limited size of these DNA molecules, replication starting from one origin can traverse the entire chromosome in a reasonable amount of time. The large linear chromosomes in eukaryotic cells, however, contain far too much DNA to be replicated speedily from a single origin. Eukaryotic replication proceeds at a rate ranging from 500 to 5000 nucleotides per minute at each replication fork (considerably slower than bacterial replication). Even at 5000 nucleotides per minute at each fork, DNA synthesis starting from a single origin would require 7 days to replicate a typical human chromosome consisting of 100 million base pairs of DNA. The replication of eukaryotic chromosomes actually takes place in a matter of minutes or hours, not days. As discussed in the introduction to the chapter, this rapid rate is possible because replication is initiated at thousands of origins. Typical eukaryotic replicons are from 20,000 to 300,000 base pairs in length. At each origin of replication, the DNA unwinds and produces a replication bubble. Replication takes place on both strands at each end of the bubble, with the two replication forks spreading outward. Eventually, the replication forks of adjacent replicons run into each other, and the replicons fuse to form long stretches of newly synthesized DNA. Replication and fusion of all the replicons leads to two identical DNA molecules. Important features of theta replication, rolling-circle replication, and linear eukaryotic replication are summarized in Table 12.2. ## 12.3 The Mechanism of Bacterial Replication Replication in bacteria begins when an **initiator protein** binds to an **origin of replication** and unwinds a short stretch of DNA, to which **DNA helicase** attaches. DNA helicase unwinds the DNA at the **replication fork**, **single-strand-binding proteins** bind to the single nucleotide strands to prevent secondary structures, and **DNA gyrase** (a topoisomerase) removes the strain ahead of the replication fork that is generated by unwinding. **During replication, primase** synthesizes short primers consisting of **RNA nucleotides**, providing a 3'-OH group to which **DNA polymerase** can add DNA nucleotides. **DNA polymerase** adds new nucleotides to the 3' end of a growing polynucleotide strand. Bacteria have two DNA polymerases that have primary roles in replication: DNA polymerase III, which synthesizes new DNA on the leading and lagging strands, and DNA polymerase I, which removes and replaces primers. **DNA ligase** seals the nicks that remain in the sugar-phosphate backbone when the RNA primers are replaced by DNA nucleotides. Several mechanisms ensure the high rate of accuracy in replication, including **precise nucleotide selection, proofreading, and mismatch repair**. ## 12.4 Eukaryotic DNA Replication Is Similar to Bacterial Replication but Differs in Several Aspects Although eukaryotic replication resembles bacterial replication in many respects, replication in eukaryotic cells presents several additional challenges. 1. The much greater size of eukaryotic genomes requires that replication be initiated at multiple origins. 2. Eukaryotic chromosomes are linear, whereas prokaryotic chromosomes are circular. 3. The DNA template is associated with histone proteins in the form of nucleosomes, and nucleosome assembly must immediately follow DNA replication. ### Eukaryotic Origins of Replication Researchers first isolated eukaryotic origins of replication from yeast cells by demonstrating that certain DNA sequences confer the ability to replicate when transferred from a yeast chromosome to small circular pieces of DNA (plasmids). These **autonomously replicating sequences (ARSs)** enabled any DNA they were attached to to replicate. They were subsequently shown to be the origins of replication in yeast chromosomes. The nature of origins of replication in many multicellular eukaryotes are still poorly understood. Origins in different eukaryotic organisms vary greatly in sequence, although they usually contain a number of A-T base pairs. Evidence suggests that eukaryotic origins are often defined more by modifications to chromatin structure (**epigenetic modifications**, see Chapter 21) than by specific sequences. A multiprotein complex, the **origin-recognition complex (ORC)**, serves as the initiator by binding to defined origins and initiating replication. In multicellular eukaryotes, there are often many more potential replication origins than are actually used during DNA synthesis. Whether the sites that are used are determined at random or are somehow specified is not known. Another difference in replication of eukaryotes involves the role of the initiator. In bacteria, helicase is unable to bind to double-stranded DNA; initiator protein first melts DNA, creating a single-stranded template that helicase then binds to. In eukaryotes, however, the initiator ORC recruits and loads helicase onto double-stranded DNA at the origin. This takes place during G₁ of the cell cycle. Then during S phase, helicase becomes activated and begins separating double-stranded DNA into single-stranded templates that are used in DNA synthesis. ### DNA Synthesis and the Cell Cycle In rapidly dividing bacteria, DNA replication is continuous. In eukaryotic cells, however, replication is coordinated with the cell cycle. Passage through the cell cycle, including the onset of replication, is controlled by cell cycle checkpoints. The important G₁/S checkpoint holds the cell in G₁ until the DNA is ready to be replicated. After the G₁/S checkpoint is passed, the cell enters S phase and the DNA is replicated. A **replication licensing system** ensures that the DNA is not replicated again until after the cell has passed through mitosis. ### The Licensing of DNA Replication As discussed in the chapter introduction, initiation of DNA replication in eukaryotes is divided into two distinct steps. 1. The origins are licensed-approved for replication. This step takes place early in the cell cycle, when **replication licensing factors** attach to each origin. 2. The replication machinery initiates replication at each licensed origin. The key is that the replication machinery functions only at licensed origins and that licensing occurs early in the cell cycle. Licensing occurs in G₁ of interphase when the **origin-recognition complex** binds to an origin. ORC, with the help of two additional licensing factors, allows a complex called MCM2-7 (for minichromosome maintenance) to bind to an origin. Then, in S phase, the MCM2-7 complex associates with several cofactors and forms an active helicase that unwinds double-stranded DNA for replication. After replication has begun, several mechanisms prevent MCM2-7 from binding to DNA and reinitiating replication at the origins until after mitosis has been completed. ### Unwinding Helicases that separate double-stranded DNA have been isolated from eukaryotic cells, as have **single-strand-binding proteins** and **topoisomerases** (which have a function equivalent to the DNA gyrase in bacterial cells). These enzymes and proteins are assumed to function in unwinding eukaryotic DNA in much the same way as their bacterial counterparts do. **Topoisomerase enzymes** are also required to remove supercoiling that builds up ahead of the replication fork in eukaryotes, just as DNA gyrase does in bacteria. Topoisomerase enzymes remove the supercoils by clamping tightly to the DNA and breaking one or both of its strands. The strands then revolve around each other, removing the supercoiling and the strain. After the DNA has relaxed, the topoisomerase reseals the broken ends of the DNA strands. Some anticancer drugs work by interfering with these topoisomerases. **Camptothecin compounds**, isolated from the happy tree *Camptotheca acuminate*, insert themselves into the DNA gap created by topoisomerase I and block the topoisomerase from resealing the broken ends of DNA. This poisons the topoisomerase so that it is unable to remove supercoils ahead of replication. Accumulating supercoils halts the replication machinery and prevents the proliferation of cancer cells. But like many other anticancer drugs, camptothecin also inhibits the replication of normal, noncancerous cells, which is why chemotherapy makes many patients sick. ### Eukaryotic DNA Polymerases Some significant differences between the processes of bacterial and eukaryotic replication are in the number and functions of DNA polymerases. Eukaryotic cells contain many more different DNA polymerases than bacteria do, which function in replication, recombination, and DNA repair. Three DNA polymerases carry out most of nuclear DNA synthesis during replication: **DNA polymerase α, DNA polymerase δ, and DNA polymerase ɛ**. - **DNA polymerase α** has primase activity and initiates nuclear DNA synthesis by synthesizing an RNA primer, followed by a short string of DNA nucleotides. After DNA polymerase α has laid down from 30 to 40 nucleotides, DNA polymerase α completes replication on the lagging strand. - **DNA polymerase δ** replicates the leading strand. - **DNA polymerase ɛ** replicates the leading strand. Other DNA polymerases take part in repair and recombination or catalyze the replication of organelle DNA. Some DNA polymerases, such as DNA polymerase δ and DNA polymerase ɛ, are capable of replicating DNA at high speed and with high fidelity (few mistakes) because they have active sites that snugly and exclusively accommodate the four normal DNA nucleotides: adenosine, guanosine, cytidine, and thymidine monophosphates. As a result of this specificity, distorted DNA templates and abnormal bases are not readily accommodated within the active site of the enzyme. When these errors are encountered in the DNA template, the high-fidelity DNA polymerases stall and are unable to bypass the lesion. Other DNA polymerases have lower fidelity but are able to bypass distortions in the DNA template. These specialized **translesion DNA polymerases** generally have a more open active site and are able to accommodate and copy templates with abnormal bases, distorted structures, and bulky lesions. Thus, these specialized enzymes can bypass such errors, but because their active sites are more open and accommodating, they tend to make more errors. In replication, high-speed, high-fidelity enzymes are generally used until they encounter a replication block. At that point, one or more of the translesion DNA polymerases takes over, bypasses the lesion, and continues replicating a short section of DNA. Then the translesion polymerases detach from the replication fork, and high-fidelity polymerases resume replication with high speed and accuracy. DNA-repair enzymes often repair the errors produced by the translesion polymerases, although some of these errors may escape detection and lead to mutations. ### Nucleosome Assembly As we have seen, eukaryotic DNA is complexed with histone proteins to form nucleosomes, structures that contribute to the stability and packing of DNA. In replication, chromatin structure is disrupted by the replication fork, but nucleosomes are quickly reassembled on the two new DNA molecules. Electron micrographs of eukaryotic DNA show that recently replicated DNA is already covered with nucleosomes. The creation of new nucleosomes requires three steps: 1. The disruption of the original nucleosomes on the parental DNA molecule ahead of the replication fork. 2. The redistribution of preexisting histones on the new DNA molecules. 3. The addition of newly synthesized histones to complete the formation of new nucleosomes. Before replication, a single DNA molecule is associated with histone proteins. After replication and nucleosome assembly, two DNA molecules are associated with histone proteins. Do the original histones of a nucleosome remain together, attached to one of the new DNA molecules, or do they disassemble and mix with new histones on both DNA molecules? Techniques similar to those employed by Meselson and Stahl to determine the mode of DNA replication were used to address this question. Cells were cultivated for several generations in a medium containing amino acids labeled with a heavy isotope. The histone proteins incorporated these heavy amino acids and were dense. The cells were then transferred to a culture medium that contained amino acids labeled with a light isotope. Histones assembled after the transfer possessed the new, light amino acids and were less dense. After replication, when the histone octamers were isolated and centrifuged in a density gradient, they formed a continuous band between the positions expected of high-density (old) and low-density (new) octamers. This finding indicates that newly assembled octamers consist of a mixture of old and new histones. Further evidence indicates that reconstituted nucleosomes appear on the new DNA molecules quickly after the new DNA emerges from the replication machinery. The reassembly of nucleosomes during replication is facilitated by proteins called **histone chaperones**, which are associated with the helicase enzyme that unwinds the DNA. The histone chaperones accept old histones from the original DNA molecule and deposit them, along with newly synthesized histones, on the two new DNA molecules. Current evidence suggests that the original histone octamer is broken down into two **H2A-H2B dimers** (each dimer consisting of one H2A and one H2B) and a single **H3-H4 tetramer** (each tetramer consisting of two H3 histones and two H4 histones). The old H3-H4 tetramer is then transferred randomly to one of the new DNA molecules and serves as a foundation onto which either new of old copies of H2A-H2B dimers are added. Newly synthesized H3-H4 tetramers and H2A-H2B dimers are also added to each new DNA molecule to complete the formation of new nucleosomes. The assembly of the new nucleosomes is facilitated by a protein called **chromatin-assembly factor 1 (CAF-1).** ### The Location of Replication Within the Nucleus The DNA polymerases that carry out replication are frequently depicted as moving down the DNA template, much as a locomotive travels along a train track. Evidence suggests that this view is incorrect. A more accurate view is that the polymerase is fixed in location and the template DNA is threaded through it, with newly synthesized DNA molecules emerging from the other end. Techniques of fluorescence microscopy, which are able to reveal active sites of DNA synthesis, show that most replication in the nucleus of a eukaryotic cell takes place at a limited number of fixed sites, often referred to as **replication factories**. Time-lapse micrographs reveal that newly duplicated DNA is extruded from these particular sites. Similar results have been obtained for bacterial cells. ### Replication at the Ends of Chromosomes A fundamental difference between eukaryotic and bacterial replication arises because eukaryotic chromosomes are linear and thus have ends. As already stated, the 3'-OH group needed by DNA polymerases is provided at the initiation of replication by RNA primers that are synthesized by primase. This solution is temporary, however, because eventually the primers must be removed and replaced by DNA nucleotides. In a circular DNA molecule, elongation around the circle eventually provides a 3'-OH group immediately in front of the primer. After the primer has been removed, the replacement DNA nucleotides can be added to this 3'-OH group. But what happens when a DNA molecule is not circular but linear? ### The End-Replication Problem In linear chromosomes with multiple origins, the elongation of DNA in adjacent replicons provides a 3'-OH group preceding each primer. At the very end of the linear chromosome, however, there is no adjacent stretch of replicated DNA to provide this crucial 3'-OH group. When the terminal primer at the end of the chromosome has been removed, it cannot be replaced by DNA nucleotides, so its removal produces a gap at the end of the chromosome, suggesting that the chromosome should become progressively shorter with each round of replication. This situation has been termed the **end-replication problem**. The end-replication problem, as originally proposed, assumed that the terminal primer is located at the very end of the chromosome. Experimental evidence suggests that in some single-celled eukaryotes, such as yeast and some protozoans, the terminal primer is indeed placed at the very end of the chromosome, but this has not been demonstrated for more complex multicellular eukaryotes. Furthermore, chromosome ends in humans are known to shorten at a much faster rate than would be expected if only the terminal primer (which is only about 10 nucleotides long) was not replaced. Research has now demonstrated that in replication of human chromosomes, the terminal primer is positioned not at the end of the chromosome but rather some 70 to 100 nucleotides from the end. This means that 70 to 100 nucleotides of DNA at the end of the chromosome are not replicated during the division of somatic cells, and the chromosome shortens by this amount each time the cell divides. ### Telomeres and Telomerase The end-replication problem suggests that chromosomes in eukaryotic cells should shorten with each cell division and eventually self-destruct. In single-celled eukaryotes, germ cells, and early embryonic cells, however, chromosomes do not shorten. So how are the ends of linear chromosomes in these cells replicated? The ends of eukaryotic chromosomes - the **telomeres** - possess several unique features, one of which is the presence of many copies of a short repeated sequence. In humans, this telomeric repeat is TTAGGG. The strand containing this G-rich repeat typically protrudes beyond the complementary C-rich strand. The single-stranded protruding end of the telomere, known as the G-rich 3' overhang, can be extended by **telomerase**, an enzyme that has both a protein and an RNA component (also known as a ribonucleoprotein). The RNA component of the enzyme contains from 15 to 22 nucleotides that are complementary to the sequence on the G-rich strand. This RNA sequence pairs with the G-rich 3' overhang and provides a template for the synthesis of additional DNA copies of the repeats. DNA nucleotides are added to the 3' end of the G-rich strand one at a time; after several nucleotides have been added, the RNA template moves down the DNA, and more nucleotides are added to the 3' end of the G-rich strand. In this way, the telomerase can extend the 3' end of the chromosome without the use of a complementary DNA template. How the complementary C-rich strand is synthesized is not clear. It may be synthesized by conventional replication, with DNA polymerase α synthesizing an RNA primer on the 5' end of the extended (G-rich) template. The removal of this primer once again leaves a gap at the 5' end of the chromosome, but this gap does not matter because the end of the chromosome is extended at each replication by telomerase, so the chromosome does not become shorter overall. Telomerase is present in single-celled eukaryotes, germ cells, early embryonic cells, and certain proliferative somatic cells (such as bone marrow cells and cells lining the intestine), all of which must undergo continuous cell division. Most somatic cells have little or no telomerase activity, and chromosomes in these cells progressively shorten with each cell division. These cells are capable of only a limited number of divisions; when the telomeres have shortened beyond a critical point, the chromosomes become unstable, have a tendency to undergo rearrangements, and are degraded. These events lead to cell death. ### Telomerase, Aging, and Disease The shortening of telomeres may contribute to the process of aging. The telomeres of genetically engineered mice that lack a functional telomerase gene (and therefore do not express telomerase in either somatic or germ cells) undergo progressive shortening in successive generations. After several generations, these mice show some signs of premature aging, such as graying, hair loss, and delayed wound healing. Through genetic engineering, it is also possible to create somatic cells that express telomerase. In these cells, telomeres do not shorten, cell aging is inhibited, and the cells divide indefinitely. Some of the strongest evidence that telomere length is related to aging comes from studies of telomeres in birds. In 2012, scientists in the United Kingdom measured telomere length in red blood cells taken from 99 zebra finches at various times during their lives. The scientists found a strong correlation between telomere length and longevity: birds with longer telomeres lived longer than birds with shorter telomeres. The strongest predictor of life span was telomere length early in life, at 25 days, which is roughly equivalent to human adolescence. Although these observations suggest that telomere length is associated with aging in some animals, the precise role of telomeres in human aging remains uncertain. Some diseases are associated with abnormalities of telomere replication. People who have **Werner syndrome**, an autosomal recessive disease, show signs of premature aging that begin in adolescence or early adulthood, including wrinkled skin, graying of the hair, baldness, cataracts, and muscle atrophy. They often develop cancer, osteoporosis, heart and artery disease, and other ailments typically associated with aging. The causative gene, WRN, has been mapped to human chromosome 8 and normally encodes a **RecQ helicase enzyme**, which is necessary for the efficient replication of telomeres. In people who have Werner syndrome, this enzyme is defective; consequently, the telomeres shorten prematurely. Another disease associated with abnormal maintenance of telomeres is **dyskeratosis congenita (DKC)**, which leads to progressive bone marrow failure, in which the bone marrow does not produce enough new blood cells. People with an X-linked form of the disease have a mutation in a gene that encodes dyskerin, a protein that normally helps process the RNA component of telomerase. People who have the disease typically inherit short telomeres from a parent who carries the mutation and who is unable to maintain telomere length in his of her germ cells owing to defective dyskerin. In families that carry this mutation, telomere length typically shortens with each successive generation, leading to anticipation: a progressive increase in severity of the disease over generations. Telomerase also appears to play a role in cancer. Cancer cells have the capacity to divide indefinitely, and telomerase is expressed in 90% of all cancers. Some recent evidence indicates that telomerase may stimulate cell proliferation independently of its effect on telomere length, so the mechanism by which telomerase contributes to cancer is not clear. As we will see in Chapter 23, cancer is a complex, multistep process that usually requires mutations in at least several genes. Telomerase activation alone does not lead to cancerous growth in most cells, but it does appear to be required, along with other mutations, for cancer to develop. Some experimental anticancer drugs work by inhibiting the action of telomerase. One of the difficulties in studying the effect of telomere shortening on the aging process is that the expression of telomerase in somatic cells also promotes cancer, which may shorten a person’s life span. To circumvent this problem, Antonia Tomás-Loba and her colleagues created genetically engineered mice that expressed telomerase and carried genes that made them resistant to cancer. These mice had longer telomeres, lived longer, and exhibited fewer age-related changes, such as skin alterations, decreases in neuromuscular coordination, and degenerative diseases, than did normal mice. These results support the idea that telomere shortening contributes to aging. ### Replication in Archaea The process of replication in archaea has a number of features in common with replication in eukaryotic cells. Many of the proteins taking part are more similar to those in eukaryotic cells than to those in bacteria. Like bacteria, some archaea have a single origin of replication, but many archaea have multiple origins, as eukaryotes do (although archaea have far fewer origins than are found in most eukaryotic chromosomes). The origins of archaea do not contain the typical sequences recognized by bacterial initiator proteins; instead, they have sequences that are similar to those found in some eukaryotic origins. The initiator proteins of archaea are also more similar to those of eukaryotes than to those of bacteria. These similarities in replication between archaeal and eukaryotic cells reinforce the conclusion that the archaea are more closely related to eukaryotic cells than to the prokaryotic bacteria. ## 12.5 Recombination Takes Place Through the Alignment, Breakage, and Repair of DNA Strands **Recombination** is the exchange of genetic information between DNA molecules; when the exchange is between homologous DNA molecules, it is called **homologous recombination**. This process takes place in crossing over, in which homologous regions of chromosomes are exchanged and alleles are shuffled into new combinations. Recombination is an extremely important genetic process because it increases genetic variation. Rates of recombination provide important information about linkage relations among genes, which is used to create genetic maps. Recombination is also essential for some types of DNA repair (as we will see in Chapter 18). Homologous recombination is a remarkable process: a nucleotide strand of one chromosome aligns precisely with a nucleotide strand of the homologous chromosome, breaks arise in corresponding regions of the two DNA molecules, parts of the molecules precisely change place, and then the pieces are correctly joined. In this complicated series of events, no genetic information is lost or gained. Although the precise molecular mechanism of homologous recombination is still not completely understood, the exchange is probably accomplished through the pairing of complementary bases. A single nucleotide strand of one chromosome pairs with the complementary strand of another, forming **heteroduplex DNA**, which is DNA consisting of nucleotide strands from different sources. In meiosis, homologous recombination (crossing over) could theoretically take place before, during, or after DNA synthesis. Cytological, biochemical, and genetic evidence indicates that it takes place in prophase I of meiosis, whereas DNA replication takes place earlier, in interphase. Thus, crossing over must entail the breaking and rejoining of chromatids when homologous chromosomes are at the four-strand stage (Figure 7.5). This section explores some theories about how the process of recombination takes place. ### Models of Recombination Homologous recombination takes place through several different pathways. One pathway is initiated by a single-strand break in each of two DNA molecules and includes the formation of a special structure called the **Holliday junction**. In this model, the double-stranded DNA molecules of two homologous chromosomes align precisely. A single-strand break in each of the DNA molecules provides a free end that invades and joins the free end of the other DNA molecule. Thus, strand invasion and joining take place on both DNA molecules, creating two heteroduplex DNAs, each consisting of one original strand plus one new strand from the other DNA molecule. The point at which the nucleotide strands pass from one DNA molecule to the other is the **Holliday junction**. The junction moves along the molecules in a process called **branch migration**. The exchange of nucleotide strands and branch migration produce a structure termed the **Holliday intermediate**, which can be cleaved in one of two ways. Cleavage in the horizontal plane, followed by rejoining of the strands, produces **noncrossover recombinants**, in which the genes on the two ends of the molecules are identical with those originally present. Cleavage in the vertical plane, followed by rejoining, produces **crossover recombinants**, in which the genes on the two ends of the molecules are different from those originally present. Another pathway for recombination is initiated by double-strand breaks in one of the two aligned DNA molecules. In this model, the removal of some nucleotides at the ends of the broken strands - followed by strand invasion, displacement, and replication - produces two **heteroduplex DNA** molecules joined by two Holliday junctions. The interconnected molecules produced in the double-strand-break model can be separated by further cleavage and reunion of the nucleotide strands in the same way that the Holliday intermediate is separated in the single-strand-break model. Whether crossover or noncrossover molecules are produced depends on whether cleavage is in the vertical or the horizontal plane. ### Enzymes Required for Recombination Recombination between DNA molecules requires the unwinding of DNA helices, the cleavage of nucleotide strands, strand invasion, and branch migration, followed by further strand cleavage and union to remove Holliday junctions. The molecular mechanism of recombination has been extensively studied in *E. coli*. Although bacteria do not undergo meiosis, they do have a type of sexual reproduction (conjugation), in which one bacterium donates its chromosome to another. Subsequent to conjugation, the recipient bacterium has two chromosomes, which may undergo homologous recombination. Geneticists have isolated mutant strains of