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Replication, Maintenance, and Rearrangements of Genomic DNA DNA Replication DNA Repair Recombination DNA Rearrangments

Replication, Maintenance, and Rearrangements of Genomic DNA DNA Replication DNA Repair Recombination DNA Rearrangments. Introduction. In order for species to evolve, mutations and gene rearrangements are needed to maintain genetic variation between individuals.

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Replication, Maintenance, and Rearrangements of Genomic DNA DNA Replication DNA Repair Recombination DNA Rearrangments

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  1. Replication, Maintenance, and Rearrangements of Genomic DNADNA ReplicationDNA RepairRecombinationDNA Rearrangments

  2. Introduction • In order for species to evolve, mutations and gene rearrangements are needed to maintain genetic variation between individuals. • DNA replication is much more complex than a single enzymatic reaction; other proteins and specific DNA sequences are also involved. • Proofreading mechanisms are required to ensure that the accuracy of replication is compatible with the low frequency of errors that is needed for cell reproduction.

  3. DNA Polymerases • DNA polymerase catalyzes the synthesis of DNA. • Cells have multiple different DNA polymerases. • All polymerases synthesize DNA only in the 5¢ to 3¢ direction. • DNA polymerases add new deoxyribonucleotides only to primer strands that are hydrogen-bonded to the parental DNA. Figure 6.1 The reaction catalyzed by DNA polymerase

  4. The Replication Fork • Growing E. coli in the presence of radioactive thymidine 3[H] initially allowed subsequent visualization of newly replicated DNA by autoradiography. • A replication fork is the region of DNA synthesis where the parental strands separate and two new daughter strands elongate. • Only one strand of DNA is synthesized in a continuous manner in the direction of overall DNA replication. Figure 6.2 Replication of E. coli DNA

  5. The Replication Fork • The leading strand is the strand of DNA that is synthesized continuously in the direction of movement of the replication fork. • The lagging strand is the strand of DNA synthesized opposite to the direction of movement of the replication fork, by ligation of Okazaki fragments. • Okazaki fragments are small pieces of newly synthesized DNA that are joined to form an intact new DNA strand. Figure 6.3 Synthesis of leading and lagging strands of DNA

  6. Initiation of Lagging Strand Synthesis • Primase is used to synthesized RNA primers. • DNA polymerase synthesizes Okazaki fragments. • DNA Ligase glues the Okazaki fragments together. Figure 6.4 Initiation of Okazaki fragments with RNA primers

  7. Lagging Strand Synthesis • Primase is an enzyme that synthesizes short fragments of RNA complementary to the lagging strand template at the replication fork. • An exonuclease is an enzyme that hydrolyzes DNA molecules in either the 5¢ to 3¢ or 3¢ to 5¢ direction. • RNase H is an enzyme that degrades the RNA strand of RNA-DNA hybrids, and 5¢ to 3¢ exonucleases.

  8. Polymerase and Replication • One class of proteins required for replication binds to DNA polymerases, increasing the activity of the polymerases and causing them to remain bound to the template DNA so that they continue synthesis of a new DNA strand. • Other proteins unwind the template DNA and stabilize single-stranded regions. Figure 6.7 Polymerase accessory proteins

  9. The Replication Fork • Helicases are enzymes that catalyze the unwinding of parental DNA. • Single-stranded DNA-binding proteins stabilize the unwound template DNA. • As the strands of parental DNA unwind, the DNA ahead of the replication fork is forced to rotate. Figure 6.8 Action of helicases and single-stranded DNA-binding proteins

  10. The Replication Fork • Topoisomerases are enzymes that catalyze the reversible breakage and rejoining of DNA strands. • The enzymes involved in DNA replication act in a coordinated manner to synthesize both leading and lagging strands of DNA simultaneously at the replication fork. Figure 6.9 Action of topoisomerases during DNA replication

  11. 6.10 Model of the E. coli replication fork • A Detailed Overview of the Replication in E.coli

  12. Origins and the Initiation of Replication • Origins of replication serve as binding sites for proteins that initiate the replication process. • Single origins are sufficient to direct the replication of bacterial and viral genomes, but multiple origins are needed to replicate the much larger genomes of eukaryotic cells within a reasonable period of time. Figure 6.12 Origin of replication in E. coli

  13. The Fidelity of Replication • The accuracy of DNA replication is critical to cell reproduction. • One mechanism by which DNA polymerase increases the fidelity of replication is by helping to select the correct base for insertion into newly synthesized DNA. • Proofreading is the selective removal of mismatched bases by DNA polymerase. • DNA polymerases require primers and catalyze the growth of DNA strands only in the 5¢ to 3¢ direction. Figure 6.11 Proofreading by DNA polymerase

  14. DNA Repair • Telomeres • DNA Damage • Mechanisms of DNA Repair

  15. Telomeres and Telomerase: Maintaining the Ends of Chromosomes • Telomeres are repeats of simple-sequence DNA that maintain the ends of linear chromosomes. • A reverse transcriptase is a DNA polymerase that uses an RNA template. • Defects in telomerase and the normal maintenance of telomeres are associated with several human diseases. Figure 6.16 Action of telomerase

  16. DNA Repair • Mutations in DNA can result from the incorporation of incorrect bases during DNA replication. • They may be: • Spontaneous • Induced by exposure to chemicals or radiation. Figure 6.17 Spontaneous damage to DNA

  17. 6.18 Examples of DNA damage induced by radiation and chemicals

  18. Direct Reversal of DNA Damage • Pyrimidine dimers are a common form of DNA damage caused by UV light in which adjacent pyrimidines are joined to form a dimer. • Photoreactivation is a process where energy derived from visible light is utilized to break the cyclobutane ring structure. • Another form of direct repair deals with damage resulting from the reaction between alkylating agents and DNA. Figure 6.19 Direct repair of thymine dimers

  19. Base Excision Repair • Base-excision repair is a process in which single damaged bases are recognized and removed from the DNA molecule. • DNA glycosylase cleaves the bond linking the base (uracil) to the deoxyribose of the DNA backbone. • AP endonuclease cleaves adjacent to AP sites in DNA. Figure 6.21 Base-excision repair

  20. Enzymes Involved in Excision Repair • Nucleotide-excision repair is a mechanism of DNA repair in which oligonucleotides containing damaged bases are removed from a DNA molecule. • Excinuclease is the protein complex that excises damaged DNA during nucleotide-excision repair in bacteria. Figure 6.22 Nucleotide-excision repair of thymine dimers

  21. 6.23 Nucleotide-excision repair in mammalian cells • Transcription-coupled repair is specifically dedicated to repairing damage within actively transcribed genes.

  22. Mismatch Excision Repair • The mismatch repair system scans newly replicated DNA and identifies and excises mismatched bases. • DNA of E. coli is modified by the methylation of adenine residues with the sequence GATC to form 6-methyladenine. Figure 6.24 Mismatch repair in E. coli

  23. Translesion DNA Synthesis • Translesion DNA synthesis provides a mechanism by which the cell can bypass DNA damage at the replication fork, which can then be corrected after replication is complete. • The enzyme polymerase V is induced in response to extensive UVA irradiation and can synthesize a new DNA strand across from a thymine dimer. Figure 6.25 Translesion DNA synthesis

  24. Recombinational Repair • Recombinational repair is a means of DNA repair that relies on replacement of the damaged DNA by recombination with an undamaged molecule. • It provides a major mechanism for repair of double strand breaks. Figure 6.26 Recombinational repair

  25. 6.27 Repair of double strand breaks

  26. Recombination between Homologous DNA Sequences • Recombination is key to the generation of genetic diversity, which is critical from the standpoint of evolution. • Homologous recombination is a molecular mechanism that involves the exchange of information between DNA molecules that share sequence homology over hundreds of bases.

  27. Models of Homologous Recombination • During recombination between homologous DNA molecules, alignment is provided by complementary base pairing strands. • Homologous recombination leads to the formation of heteroduplex regions. Figure 6.28 Homologous recombination by complementary base pairing

  28. 6.29 The Holliday model for homologous recombination • The Holliday model is a molecular model of genetic recombination involving the formation of heteroduplex regions.

  29. DNA Rearrangements • Several types of DNA rearrangements are now recognized in both prokaryotic and eukaryotic cells. • Transposable elements constitute a large fraction of the genomes of plants and animals, including nearly half of the human genome. • Site-specific recombination is mediated by proteins that recognize specific DNA sequences, such as antibodies or cell receptors

  30. DNA Rearrangements • Immunoglobulins consist of pairs of identical heavy and light polypeptide chains. • The genes that encode immunoglobulin light chains consist of three regions: a V region, a joining (J) region, and a C region. • Heavy-chain genes include a fourth region known as the diversity, or D, region, which encodes amino acids lying between V and J. Figure 6.36 Structure of an immunoglobulin

  31. 6.38 Rearrangement of immunoglobulin heavy-chain genes • DNA rearrangements are initiated by introducing a double strand break between the recombination signal sequences and the coding sequences.

  32. Site-Specific Recombination • Class switch recombination is a type of region-specific recombination responsible for the association of rearranged immunoglobulin V(D)J regions with different heavy chain constant regions. • Class switch recombination transfers a rearranged variable region to a new downstream constant region, with deletion of the intervening DNA. Figure 6.41 Class switch recombination

  33. Transposition via DNA Intermediates • Transposable elements, or transposons, are DNA sequences that can move to different positions in the genome. • The first transposons that were characterized in bacteria, which move via DNA intermediates. Figure 6.43 Bacterial transposons

  34. Transposition via RNA Intermediates • Retrotransposons are transposable elements that move via reverse transcription of an RNA intermediate. • Retroviruses contain RNA genomes in their virus particles but replicate via the synthesis of a DNA provirus. • Reverse transcriptase is a DNA polymerase that uses an RNA template. Figure 6.44 The organization of retroviral DNA

  35. DNA Amplification • Additional copies of genes can result from the replication process. • Gene amplification occurs as an abnormal event in cancer cells

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