Chapter 11 DNA Replication and Recombination PDF

Chapter 11 DNA Replication and Recombination Genetic continuity is maintained by semiconservative replication of DNA, where each strand of the parent double helix serves as a template for a new strand. Semiconservative replication results in each new double helix containing one old...

Chapter 11 DNA Replication and Recombination Genetic continuity is maintained by semiconservative replication of DNA, where each strand of the parent double helix serves as a template for a new strand. Semiconservative replication results in each new double helix containing one old strand and one new strand of DNA. DNA synthesis is directed by various enzymes and proteins, making it a complex but orderly process. The process involves the polymerization of nucleotides into polynucleotide chains. While DNA synthesis is fundamentally similar in bacteria and eukaryotes, it is more complex in eukaryotes. In eukaryotes, the synthesis of DNA at chromosome ends (telomeres) is facilitated by the enzyme telomerase, which contains RNA. Genetic recombination allows for the exchange of genetic information between DNA molecules. Learning Objectives DNA replication is semiconservative, meaning each new DNA molecule consists of one old strand and one new strand. This was supported by experiments on viruses, bacteria, and eukaryotes. DNA polymerase I, II, and III have distinct roles in bacterial DNA replication. DNA polymerase III is the primary enzyme for DNA synthesis, while DNA polymerase I is involved in removing RNA primers and filling in gaps, and DNA polymerase II is involved in DNA repair. DNA polymerase III consists of nine subunits, each with specific functions essential for DNA replication. DNA synthesis can be continuous on the leading strand and discontinuous on the lagging strand, resulting in Okazaki fragments. DNA replication in bacteria involves initiation at the origin of replication, elongation by DNA polymerase, and proofreading to correct errors. The rate and duration of DNA synthesis can be used to calculate the number of base pairs and the physical length of a bacterial chromosome. Mutations affecting DNA replication can be predicted based on their impact on the replication process. DNA replication in eukaryotes is more complex than in bacteria, involving multiple origins of replication and additional regulatory mechanisms. Telomeres pose a challenge for replication due to their linear nature, but telomerase extends telomeres to prevent loss of genetic information. The Holliday structure is a key intermediate in genetic recombination, and its resolution is crucial for accurate DNA repair and recombination. 11.1 DNA Is Reproduced by Semiconservative Replication Watson and Crick proposed that each strand of a DNA double helix can serve as a template for synthesizing its complementary strand. When the helix unwinds, each nucleotide on the parent strands attracts its complementary nucleotide due to potential hydrogen bonds. Thymidylic acid (T) pairs with adenylic acid (A), and guanidylic acid (G) pairs with cytidylic acid (C). Covalent linking of these nucleotides into polynucleotide chains along both templates results in two identical double strands of DNA. Each replicated DNA molecule consists of one old strand and one new strand, a process known as semiconservative replication. The semiconservative mode of DNA replication is supported by experimental results. In the first replication cycle with a radioactive isotope, both sister chromatids show radioactivity, indicating each contains one new radioactive DNA strand and one old unlabeled strand.  In the second replication cycle in an unlabeled medium, only one of the two sister chromatids of each chromosome is radioactive, as half of the parent strands are unlabeled. The Meselson–Stahl experiment and the experiment by Taylor, Woods, and Hughes confirmed the semiconservative replication model. Subsequent studies with other organisms also supported the semiconservative replication model and Watson and Crick’s double-helix DNA model. Origins, Forks, and Units of Replication DNA replication begins at specific regions called origins of replication. In bacteria like E. coli, there is a single origin known as oriC. Replication can be unidirectional or bidirectional. In E. coli, replication is bidirectional, meaning it proceeds in both directions from the origin. The replication fork is the structure formed when the DNA helix is unwound at the origin. In bidirectional replication, two replication forks move in opposite directions. A replicon is the length of DNA replicated from a single origin. In E. coli, the entire chromosome, which is 4.6 million base pairs, acts as one replicon. John Cairns used radioactive DNA precursors and autoradiography to show that E. coli has a single origin of replication. 11.2 DNA Synthesis in Bacteria Involves Five Polymerases, as Well as Other Enzymes Replication is semiconservative and bidirectional, meaning each new DNA molecule consists of one old strand and one new strand, and replication proceeds in two directions from the origin. The synthesis of long complementary polynucleotide chains on a DNA template is a complex process. Initial studies on DNA synthesis were conducted using microorganisms, coinciding with the Meselson– Stahl experiment. DNA synthesis remains a highly complex and actively researched area in molecular biology. DNA Polymerase I DNA polymerase I was first isolated by Arthur Kornberg and colleagues in 1957 from E. coli, enabling DNA synthesis in vitro. Two major requirements for DNA synthesis by DNA polymerase I are all four deoxyribonucleoside triphosphates (dNTPs) and template DNA. Omission of any dNTP or substitution with nucleotides or nucleoside diphosphates results in no measurable DNA synthesis. Absence of template DNA leads to significantly reduced DNA synthesis. The enzyme DNA polymerase I is a single polypeptide with 928 amino acids and facilitates semiconservative replication. DNA polymerase I adds nucleotides to the growing DNA chain with high specificity. The precursor dNTP has three phosphate groups attached to the 5' carbon of deoxyribose. During DNA synthesis, the two terminal phosphates are cleaved, leaving one phosphate attached to the 5' carbon. This remaining phosphate forms a covalent bond with the 3'-OH group of the deoxyribose on the growing chain. DNA chain elongation occurs in the 5' to 3' direction, adding one nucleotide at a time to the 3' end. Each addition exposes a new 3'-OH group for the next nucleotide to attach. DNA Polymerase II, III, IV, and V DNA polymerase I (DNA Pol I) directs DNA synthesis but its true biological role was questioned in 1969. Paula DeLucia and John Cairns discovered a mutant E. coli strain (polA1) deficient in DNA Pol I activity, which could still replicate DNA and reproduce but had impaired DNA repair capabilities. The mutant strain was highly sensitive to UV light and radiation, indicating DNA Pol I's role in DNA repair. Two conclusions were drawn: another enzyme replicates DNA in vivo in E. coli, and DNA Pol I is crucial for maintaining DNA synthesis fidelity. Four other unique DNA polymerases have been identified in cells lacking DNA Pol I and in normal cells. DNA Pol I, II, and III cannot initiate DNA synthesis but can elongate an existing DNA strand (primer) and possess 3'→5' exonuclease activity. This exonuclease activity allows them to polymerize in one direction, reverse, and excise incorrect nucleotides, providing a proofreading function. Properties of Bacterial DNA Polymerases I, II, and III DNA Pol I, II, and III all exhibit 5'→3' polymerization and 3'→5' exonuclease activity, but only DNA Pol I has 5'→3' exonuclease activity. DNA Pol I is abundant and stable, excising nucleotides in the same direction as synthesis, and is involved in primer removal and gap-filling synthesis. DNA Pol III is the primary enzyme for 5'→3' polymerization during in vivo replication and has a proofreading function via its 3'→5' exonuclease activity. DNA Pol II, IV, and V are primarily involved in DNA repair, particularly in response to damage from external sources like ultraviolet light. The DNA Pol III Holoenzyme The DNA Pol III holoenzyme is a complex molecule composed of multiple polypeptide subunits, with ten identified subunits. The core enzyme complex, consisting of the α, ε, and θ subunits, is responsible for the catalytic function of the holoenzyme. - The α subunit is responsible for DNA synthesis. - The ε subunit has 3' to 5' exonuclease activity, which is crucial for proofreading. Each holoenzyme in E. coli contains two or possibly three core enzyme complexes. The sliding clamp loader, composed of the γ, δ, δ′, χ, and ν subunits, pairs with the core enzyme and facilitates the function of the sliding DNA clamp. - The sliding clamp loader's function depends on ATP hydrolysis. The sliding DNA clamp, made up of multiple β subunits, encircles the DNA helix and maintains the core enzyme's attachment to the template during nucleotide polymerization, increasing processivity. Each core enzyme is linked to one sliding clamp, and the τ subunit connects each core enzyme to the sliding clamp loader. 11.3 Many Complex Issues Must Be Resolved during DNA Replication DNA replication in bacteria and viruses is semiconservative and bidirectional, occurring along a single replicon. DNA synthesis is catalyzed by DNA polymerase III and proceeds in the 5' to 3' direction.  Bidirectional synthesis creates two replication forks that move in opposite directions from the origin. The DNA helix must undergo localized unwinding, and the open configuration must be stabilized for synthesis to proceed. Unwinding and synthesis increase coiling tension further down the helix, which must be reduced. RNA, not DNA, serves as the primer for DNA polymerase III to commence polymerization. DNA polymerase III synthesizes the DNA complement of both strands, with continuous synthesis on one strand and discontinuous synthesis on the antiparallel strand. RNA primers must be removed, and the resulting gaps filled with DNA complementary to the template. The newly synthesized DNA must be joined to adjacent strands. DNA polymerases have a proofreading mechanism to correct errors, ensuring accurate replication. Unwinding the DNA Helix DNA replication in most bacteria and viruses starts at a single origin point on the circular chromosome, known as oriC in E. coli. oriC consists of 245 DNA base pairs, including five 9 base pair repeats (9mers) and three 13 base pair repeats (13mers), both of which are AT-rich and less stable, aiding in helical unwinding. The initiator protein DnaA binds to the 9mers, causing a conformational change that allows it to associate with the 13mers, leading to helix destabilization and exposure of single-stranded DNA (ssDNA). DNA helicase, composed of DnaB polypeptides, assembles around the ssDNA and recruits the holoenzyme to the replication fork, initiating replication. Helicases use ATP hydrolysis to provide the energy needed to denature the hydrogen bonds stabilizing the double helix, allowing the helicase to move along the ssDNA and continue unwinding the helix. Single-stranded binding proteins (SSBs) bind to single strands of DNA to prevent base pairing until the DNA can serve as a template for synthesis. As the DNA helix unwinds, coiling tension is created ahead of the replication fork, leading to supercoiling. Supercoiling in circular DNA molecules can be visualized as added twists and turns, similar to twisting a stretched rubber band. DNA gyrase, a type of DNA topoisomerase, relaxes supercoiling by making single- or double-stranded cuts and catalyzing movements to undo twists and knots. The energy for these reactions is provided by ATP hydrolysis. The replisome is a complex of DNA, polymerase, and associated enzymes that initiate DNA synthesis. DNA polymerase III requires a primer with a free 3′-hydroxyl group to elongate a polynucleotide chain. RNA serves as the primer for initiating DNA synthesis, as no free 3′-hydroxyl group is available in a circular chromosome. A short RNA segment (10-12 nucleotides) complementary to DNA is synthesized on the DNA template. Primase, a form of RNA polymerase, synthesizes the RNA primer and is recruited to the replication fork by DNA helicase. Primase does not require a free 3′ end to initiate synthesis. DNA Pol III adds deoxyribonucleotides to the RNA primer, initiating DNA synthesis. The RNA primer is later removed and replaced with DNA, likely under the direction of DNA Pol I. RNA priming is a universal phenomenon observed in viruses, bacteria, and several eukaryotic organisms during DNA synthesis initiation. The two strands of a DNA double helix are antiparallel, meaning one strand runs in the 5′ to 3′ direction, while the other runs in the 3′ to 5′ direction. DNA polymerase III (DNA Pol III) synthesizes DNA only in the 5′ to 3′ direction. The donut-shaped sliding DNA clamp surrounds the unreplicated double helix and is linked to the advancing core enzyme. The clamp prevents the core enzyme from dissociating from the template during polymerization. This prevention increases the processivity of the core enzyme, allowing more nucleotides to be added continuously before dissociation.  The increased processivity is crucial for the rapid in vivo rate of DNA synthesis during replication. During replication, the leading strand is synthesized continuously in the same direction as the replication fork movement. The lagging strand is synthesized discontinuously in the opposite direction of the replication fork movement, requiring multiple points of initiation. The discontinuous segments of the lagging strand are known as Okazaki fragments. Reiji and Tuneko Okazaki discovered that DNA replication in E. coli involves small fragments of newly formed DNA, now known as Okazaki fragments, which are 1000 to 2000 nucleotides long. Okazaki fragments are initially bonded to the template strand and contain RNA primers. These fragments are converted into longer DNA strands as synthesis progresses. Discontinuous DNA synthesis requires enzymes to remove RNA primers and join Okazaki fragments into the lagging strand. DNA Polymerase I (DNA Pol I) removes RNA primers and replaces them with DNA nucleotides. DNA ligase catalyzes the formation of phosphodiester bonds to seal nicks between Okazaki fragments, joining them into a continuous strand. The role of DNA ligase is confirmed by the accumulation of unjoined Okazaki fragments in a ligase- deficient mutant strain of E. coli. Concurrent Synthesis Occurs on the Leading and Lagging Strands Both the leading and lagging strands of DNA are replicated simultaneously at the same replication fork by the DNA Pol III holoenzyme. Each strand is acted upon by one of the two core enzymes that are part of the DNA Pol III holoenzyme. The lagging strand forms a loop, allowing nucleotide polymerization to occur simultaneously on both template strands. After synthesizing 1000 to 2000 nucleotides, the enzyme on the lagging strand encounters a completed Okazaki fragment and releases the lagging strand. A new loop of the lagging strand is then spooled out, and the process repeats. Looping inverts the orientation of the template but maintains the synthesis direction on the lagging strand in the 5' to 3' direction. There is believed to be a third core enzyme associated with the DNA Pol III holoenzyme that functions in the synthesis of Okazaki fragments. Proofreading and Error Correction Occurs during DNA Replication DNA replication aims to synthesize a new strand that is complementary to the template strand at each nucleotide position. DNA polymerases are highly accurate but occasionally insert noncomplementary nucleotides. DNA polymerases possess 3' to 5' exonuclease activity, allowing them to detect and excise mismatched nucleotides. After removing a mismatched nucleotide, 5' to 3' synthesis resumes. This proofreading process increases the fidelity of DNA synthesis by about 100 times. In DNA polymerase III, the epsilon (ε) subunit of the core enzyme is involved in proofreading. Mutations in the ε subunit in E. coli lead to a significantly higher error rate during DNA synthesis. 11.4 A Coherent Model Summarizes DNA Replication  At the replication fork, helicase unwinds the double helix, and single-stranded binding proteins stabilize the unwound strands. DNA gyrase reduces the tension from supercoiling ahead of the replication fork. DNA Pol III holoenzyme, with its core enzymes attached to the template strands by sliding DNA clamps, facilitates replication. Continuous synthesis occurs on the leading strand, while the lagging strand loops to allow concurrent synthesis. DNA polymerase I replaces RNA primers with DNA on the lagging strand. 11.6 Eukaryotic DNA Replication Is Similar to Replication in Bacteria, but Is More Complex Eukaryotic DNA replication shares core mechanisms with bacterial replication, including the unwinding of double-stranded DNA at replication origins, formation of replication forks, and bidirectional synthesis of leading and lagging strands. Both eukaryotic and bacterial DNA polymerases require four deoxyribonucleoside triphosphates, a template, and a primer for DNA synthesis. Eukaryotic DNA replication is more complex due to the larger amount of DNA, the presence of nucleosomes, and the linear structure of eukaryotic chromosomes. Initiation at Multiple Replication Origins Eukaryotic DNA replication involves managing significantly larger amounts of DNA compared to bacterial replication. For instance, yeast cells have 3 times more DNA, and Drosophila cells have 40 times more DNA than E. coli cells. Eukaryotic DNA polymerases synthesize DNA much slower than bacterial DNA polymerases, at a rate of about 2000 nucleotides per minute, which is 25 times slower. To complete replication efficiently, eukaryotic chromosomes have multiple replication origins. Yeast genomes have between 250 and 400 origins, while mammalian genomes can have up to 25,000. These multiple origins create "replication bubbles" visible under an electron microscope, each bubble providing two replication forks. In yeast, replication origins are known as autonomously replicating sequences (ARSs) and consist of about 120 base pairs with a consensus sequence of 11 base pairs. In mammalian cells, replication origins are not defined by specific sequence motifs but rather by chromatin structure over regions of 6–55 kb. Multiple Eukaryotic DNA Polymerases Eukaryotic cells have significantly more DNA polymerase molecules compared to bacterial cells to manage their numerous replicons. A single E. coli cell has about 15 DNA polymerase III molecules, whereas a mammalian cell has tens of thousands of DNA polymerase molecules. Eukaryotes use a greater variety of DNA polymerase types than bacteria. The human genome encodes at least 14 different DNA polymerases, with only three primarily responsible for most nuclear genome DNA replication. Properties of Eukaryotic DNA Polymerases  DNA polymerases have specific functions: Pol α initiates DNA synthesis with RNA/DNA primers, Pol δ synthesizes the lagging strand and participates in repair and recombination, and Pol ε synthesizes the leading strand and also participates in repair and recombination. Pol α has low processivity, meaning it synthesizes short DNA segments before dissociating. It is replaced by Pol δ or Pol ε, which have higher processivity and proofreading capabilities (3'→5' exonuclease activity). Pol ε synthesizes the leading strand, while Pol δ synthesizes the lagging strand. Both are essential for DNA replication and repair. Eukaryotic Okazaki fragments are smaller (100-150 nucleotides) compared to bacterial ones. Pol γ is responsible for mitochondrial DNA replication and repair. Other polymerases (η, ζ, κ, ν, ι, θ, λ, μ, Rev1) are involved in DNA synthesis and repair, allowing replication through damaged DNA regions but with higher error rates. Replication through Chromatin Eukaryotic DNA is complexed with DNA-binding proteins and exists as chromatin. Chromatin is composed of nucleosomes, which are units of about 200 base pairs of DNA wrapped around eight histone proteins. Before DNA polymerases can synthesize DNA, nucleosomes and other DNA-binding proteins must be removed or modified. During DNA synthesis, histones and nonhistone proteins rapidly reassociate with the new DNA duplexes to reestablish nucleosome patterns. Electron microscopy studies show that nucleosomes form immediately after new DNA is synthesized at replication forks. Nucleosomes are disrupted just ahead of the replication fork. Preexisting histone proteins combine with newly synthesized histone proteins to form new nucleosomes. New nucleosomes are assembled behind the replication fork onto the two daughter strands of DNA. Chromatin assembly factors (CAFs) facilitate the assembly of new nucleosomes and move along with the replication fork. 11.7 Telomeres Solve Stability and Replication Problems at Eukaryotic Chromosome Ends Bacterial DNA is circular, while eukaryotic DNA is linear, leading to different challenges in DNA synthesis. Linear DNA ends in eukaryotic chromosomes resemble double-stranded breaks (DSBs), which can trigger DNA repair mechanisms, potentially causing chromosome fusions and translocations. If linear DNA ends do not fuse, they are susceptible to degradation by nucleases. DNA polymerases cannot synthesize new DNA at the tips of single-stranded 5' ends, posing a replication problem. Eukaryotic chromosomes have telomeres at their ends to address these issues. Telomere Structure and Chromosome Stability In 1978, Elizabeth Blackburn and Joe Gall discovered unique structures at the ends of chromosomes in Tetrahymena, consisting of the sequence 5'−⊤GGGG−3' repeated 30 to 60 times, known as the G-rich strand, with a complementary C-rich strand 5'−∀CC−3'  Similar tandemly repeated DNA sequences, called telomeres, are found at the ends of linear chromosomes in most eukaryotes. In humans, the telomeric sequence 5'−⊤AGGG−3' is repeated thousands of times, with telomere lengths ranging from 5 to 15 kb. Telomere lengths vary among species: yeast telomeres are several hundred base pairs long, while mouse telomeres range from 20 to 50 kb. Each linear chromosome ends with two antiparallel DNA strands, with the G-rich strand ending at the 3' end, which is significant for telomere replication. Telomeric DNA has two key features for protecting chromosome ends: - A single-stranded DNA tail extends from the 3' G-rich strand, varying in length between organisms (12-16 nucleotides in Tetrahymena, 30-400 nucleotides in mammals). - The 3' ends of these tails can form loop structures (t-loops) by interacting with upstream sequences, resembling shoelace bows. Telomeres and Chromosome End Replication DNA replication starts from short RNA primers on both leading and lagging strands because DNA polymerase needs a free 3'−OH to begin synthesis. After replication, RNA primers are removed, and the resulting gaps in the daughter strands are filled by DNA polymerase and sealed by ligase. Internal gaps in the new DNA strands have free 3'−OH groups at the ends of Okazaki fragments, allowing DNA polymerase to initiate synthesis. A problem occurs at the 5' ends of the newly synthesized DNA because these gaps lack free 3'−OH groups, preventing DNA polymerase from filling them. During DNA replication, gaps remain on newly synthesized DNA strands due to the removal of RNA primers, leading to the progressive shortening of chromosome ends. This shortening can eventually affect gene-coding regions if it extends beyond the telomere. Telomerase, a unique eukaryotic enzyme, solves this end-replication problem by adding repeated nucleotide sequences to the ends of chromosomes. Telomerase was discovered by Elizabeth Blackburn and Carol Greider in Tetrahymena, where the telomeric DNA sequence is 5'−⊤GGGG−3'. Telomerase is a ribonucleoprotein that includes an RNA component (TERC) and a catalytic subunit (TERT). TERC serves as a guide for enzyme attachment to the telomere and as a template for DNA synthesis, a process known as reverse transcription. TERT is the catalytic subunit responsible for the reverse transcription activity. In Tetrahymena, TERC contains the sequence C∀CC∀, which complements the telomeric DNA sequence ⊤GGGG. The enzyme extends the 3' tail of the G-rich strand by synthesizing DNA using the TERC RNA as a template. The enzyme likely translocates to the new end of the tail and repeats the extension process. After the telomere 3' tail is lengthened by telomerase, conventional DNA synthesis begins. Primase lays down a primer near the end of the telomere tail. DNA polymerase and ligase fill most of the gap. A small gap remains when the primer is removed, but it is beyond the original end of the chromosome, preventing chromosome shortening. Telomerase function is present in all studied eukaryotes. Telomeric DNA sequences are highly conserved throughout evolution, indicating their critical function. Telomeres in Disease, Aging, and Cancer Only certain cell types, such as embryonic stem cells, some adult stem cells, epidermal cells, and immune cells, express telomerase, which maintains telomere length.  Most other cells do not express telomerase, leading to telomere erosion, chromosome damage, and cellular senescence after many divisions. Rare diseases like dyskeratosis congenita are linked to mutations in telomerase or shelterin subunits, causing symptoms of premature aging and early death due to stem cell failure. Premature aging syndromes like Fanconi anemia and Werner syndrome are associated with short telomeres but involve mutations in DNA damage repair genes, not telomere maintenance. There is a correlation between telomere length/telomerase activity and common diseases such as diabetes and heart disease. Telomere shortening is linked to aging, as critically short telomeres cause cells to enter senescence, leading to less efficient cell function and physiological aging changes. Most healthy somatic cells lack telomerase activity, leading to telomere shortening and senescence, while over 90% of cancer cells maintain telomere length through telomerase or alternative lengthening of telomeres (ALT). Cancer cells can be created in tissue cultures by introducing telomerase activity along with mutations in proto-oncogenes and tumor-suppressor genes. The necessity of telomerase in cancer cells suggests potential for anti-telomerase drugs to inhibit tumor growth, with some drugs already in phase III clinical trials. Genetics, Ethics, and Society Human cells have a finite life span, with normal human fibroblasts becoming senescent after about 50 cell divisions. Cellular senescence may be linked to aging, as cells from younger individuals and longer-lived species undergo more divisions than those from older individuals and shorter-lived species. Telomeres, the ends of chromosomes, shorten with each DNA replication in most mammalian somatic cells due to the lack of telomerase activity. Epidemiological studies show correlations between telomere length and life span, as well as between shorter telomeres and common diseases or lifestyle factors like smoking, poor diets, and stress. The relationship between telomere length and longevity in humans is debated and inconsistent. Telomerase activity has been linked to aging; increasing telomerase activity in human cells in culture extends their growth beyond typical senescence. Mice with defective telomerase (TERT subunit) exhibit symptoms of aging, which can be reversed by reactivating telomerase, leading to increased life spans. Overexpression of telomerase in normal mice has been associated with increased life spans, though it is unclear if telomere lengths were altered. The potential for reversing aging symptoms in humans by activating telomerase genes is suggested, but the direct causative role of telomerase activation or telomere lengthening remains debated.