DNA is a polymer composed of deoxynucleoside monophosphates that are connected by 3′→5′-phosphodiester bonds (Fig. 30.32). This results in a long, unbranched chain that has polarity, with a 5′-end (free phosphate) and a 3′-end (free hydroxyl). The sequence of nucleotides is read in the 5′→3′ direction. DNA exists as a double-stranded molecule, where the two chains are paired in an antiparallel manner and twist around each other to form a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix acts as a template for building a complementary daughter strand (semiconservative replication). DNA replication occurs during the S phase of the cell cycle and begins at a specific origin of replication. As the two strands unwind and separate, synthesis takes place at two replication forks that move in opposite directions away from the origin (bidirectionally). The enzyme helicase unwinds the double helix. As the two strands separate, positive supercoils form ahead of the replication fork, while negative supercoils form behind the fork. DNA topoisomerases types I and II remove the supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. This means that one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand), while the other grows in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, which is a short stretch of RNA synthesized by primase. Continuous synthesis on the leading strand only needs one RNA primer, whereas discontinuous synthesis on the lagging strand involves the formation of multiple RNA primers and Okazaki fragments. In Escherichia coli (E. coli), DNA chain elongation is carried out by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. This enzyme also proofreads the newly synthesized DNA by removing mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, which has a 5′→3′ exonuclease activity. DNA pol I fills in the gaps with DNA and performs proofreading during synthesis. The final phosphodiester linkage is formed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, with one of its subunits being a primase. Pol α has a 5′→3′ polymerase activity that adds a short piece of DNA to the RNA primer. Pol ε is responsible for completing DNA synthesis on the leading strand, while pol δ elongates each fragment of the lagging strand. Pol β is involved in DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity for proofreading. Nucleoside analogs containing modified sugars can be used to inhibit DNA chain growth and are useful in anticancer and antiviral chemotherapy. Telomeres are regions of highly repetitive DNA that are associated with proteins and protect the ends of linear chromosomes. As cells divide and age, these sequences become shorter, contributing to cellular senescence. However, in cells that do not undergo senescence, such as germline and cancer cells, the ribonucleoprotein telomerase uses its reverse transcriptase component to extend the telomeres, with its RNA component serving as a template. There are five classes of positively charged histone (H) proteins. Two copies each of histones H2A, H2B, H3, and H4 form an octameric core structure around which DNA is wrapped, creating a nucleosome. The DNA that connects the nucleosomes, known as linker DNA, is bound to H1. Nucleosomes can be tightly packed together to form a nucleofilament, and further levels of organization result in the formation of a chromosome. Most DNA damage can be repaired through excision repair, which involves the recognition and removal of damaged regions by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers, which are recognized and removed in E. coli by the uvrABC proteins involved in nucleotide excision repair. Defects in the XP proteins, necessary for nucleotide excision repair of thymine dimers in humans, lead to xeroderma pigmentosum. Mismatched bases are repaired in a similar way through recognition and removal by Mut proteins in E. coli. The extent of methylation is used to identify the strands in prokaryotes. In humans, defective mismatch repair by homologous proteins is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases, such as uracil, are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the site of damage is then cut out. Double-strand breaks in DNA are repaired through nonhomologous end joining, which is error-prone, or template-requiring homologous recombination, which is considered "error-free."
DNA is a polymer of deoxynucleoside monophosphates covalently linked by 3′→5′-phosphodiester bonds (Fig. 30.32). The resulting long, unbranched chain has polarity, with both a 5′-end (free phosphate) and a 3′end (free hydroxyl). The sequence of nucleotides is read 5′→3′. DNA exists as a double-stranded molecule, in which the two chains are paired in an antiparallel manner and wind around each other, forming a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at an origin of replication. As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork and negative supercoils behind the fork. DNA topoisomerases types I and II remove supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand) and one in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, a short stretch of RNA synthesized by primase. Leading-strand synthesis needs only one RNA primer (continuous synthesis), whereas the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. The enzyme proofreads the newly synthesized DNA, removing terminal mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, using its 5′→3′ exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, one subunit of which is a primase. Pol α 5′→3′ polymerase activity adds a short piece of DNA to the RNA primer. Pol ε completes DNA synthesis on the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”).