DNA Replication, Repair, and Recombination PDF

DNA Replication, Repair, and Recombination Chapter 25 Objectives } DNA Damage } DNA Repair Mechanisms } Recombination } General/Homologous Recombination } CRISPR-Cas 2 PHSC 208 Topic 25 2/19/...

DNA Replication, Repair, and Recombination Chapter 25 Objectives } DNA Damage } DNA Repair Mechanisms } Recombination } General/Homologous Recombination } CRISPR-Cas 2 PHSC 208 Topic 25 2/19/2025  DNA Damage § Mutations are caused by metabolic activities or environmental exposures on DNA § The natural rate of mutation is about 1.0 mutation per 100,000 genes per generation (germline, higher eukaryotes) } Somatic cell mutations less problematic but can be a cause of cancer } Environmental and chemical agents can damage DNA to generate mutations } UV light } Ionizing radiation } Can generate hydroxyl radical } Reactive chemicals Figure 25-30 Intrastrand } Spontaneous purine hydrolysis } 20,000 of 6 billion per day Thymine Dimer 3 PHSC 208 Topic 25.4 2/19/2025 Types of DNA Mutations } DNA is designed for information storage but it is not a static molecule } Point Mutations—single base change, single nucleotide polymorphisms (SNPs) } Transitions: Base is replaced with the same type of base (Py or Pu) } Transversions: Purine replaces pyrimidine (or vice versa) } SNP classification if within protein coding sequence } Silent mutation-no change in protein sequence } Nonsense mutation-premature stop of protein synthesis } Missense mutation-alters protein sequence } Indels—Insertions or Deletions } Generally involve more than one base (1-1000’s) } Frameshift mutation-indel in protein coding sequence, if not a multiple of 3 4 PHSC 208 Topic 25.4 2/19/2025  Altered Bases from Chemical Damage } Nitrous acid can oxidatively deaminate bases } Can cause both A.T to G.C and G.C to A.T transitions } Reactive oxygen species are part of normal cell metabolism } Superoxide, hydroxy radicals, peroxide } 8-oxoguanine, e.g. ¨ Can yield a G.C to T.A transversion } Alkylation of purines at N7 position promotes hydrolysis of the glycosidic bond } Repair system is error prone DNA Alkylating Reagents } Spontaneous hydrolysis of purines about 20,000 of 6 billion per day 5 PHSC 208 Topic 17.1A 2/19/2020  Ames Test for Mutagenesis } Many mutagens are also carcinogens } Standard animal tests for carcinogenesis are expensive and can take 3 years } Ames test is a simple test for mutagenesis } Salmonella typhimurium ¨ His- ¨ Cannot grow unless media is supplemented with histidine ¨ If a correct mutation arises, can become his+ ¨ 109 bacteria plated and wait for 2 days 6 PHSC 208 Topic 25.4 2/19/2025  DNA Repair } Typical mammalian cell has about 100,000 molecular lesions to its DNA per day } Importance of repair is underscored by the variety of repair mechanisms in cells } Direct Reversal of Damage } The enzyme photolyase uses light to excite electrons in cyclobutane ring catalyzing the retro-Diels-Alder reaction } Separates adjacent linked pyrimidine rings by “base flipping” Figure 25-33: DNA photolyase } Placental mammals lack photolyase } O6-alkylguanine-DNA alkyltransferase ¨ Removes methyl group with a Cys residue ¨ Permanent enzyme methylation 7 PHSC 208 Topic 25.5 2/19/2025 Base Excision Repair } DNA glycosylases cleave the N- glycoside linkage of a damaged base and the deoxyribose } 8-oxoguanine and uracil in DNA } Uracil-DNA glycosylase (UDG) recognizes U-G mismatch and Figure 25-34 excises } Apurinic or apyrimidinic sites are resolved through the action of nucleases that remove the residue, DNA polymerase (pol I in bacteria; DNA polymerase b in mammals), and DNA ligase Figure 25-35. human UDG 8 PHSC 208 Topic 25.5 2/19/2025  Nucleotide Excision Repair } Contained by all cells } Corrects pyrimidine dimers and other bulky lesions that distort the bases from their normal positions } UvrABC endonuclease system in E. coli } Two cuts are made in the DNA in the damaged strand, one on either side of the damage. } E. coli excision nucleases Uvr A, B, and C } Excised DNA of 11-12 bases is removed by UvrD helicase } The gap is filled, and the nick ligated. } Xeroderma Pigmentosum Figure 25-36 } Disorder where patients are deficient in one of the activities needed for this kind of repair. } Extreme sensitivity to light 9 PHSC 208 Topic 18.1 2/19/2025  Mismatch Repair } Single-strand repair mechanism that correct helix distorting base mispairings } Proofreading errors } Replication slippage } Errors that escaped editing functions during transcription } Key feature is capacity to distinguish between old and newly synthesized strands } In prokaryotes, methylation marks template strand } MutS homodimer recognizes mismatches } Recruits additional proteins to selectively cleave out portion of new strand } Eukaryotes have homologs of MutS and L may use lagging strand status (unsealed nicks) to mark new strand 10 PHSC 208 Topic 25.5 2/19/2025 Figure 25-37 Double Strand Break Repair } Double strand breaks occur with inter- strand cross-links, topoisomerase inhibition/deficiency, and with ionizing radiation damage. } 5-10% of dividing cells in culture exhibit a chromosomal break } Two pathways for repair } Non-homologous end joining (NHEJ) } Removes or extends ssDNA and brings two ends together for ligation ¨ Core of end-joining complex is the Figure 25-38. Ku protein with 14-bp of DNA protein Ku } Error prone due to no requirement for sequence homology } Homologous Recombination 11 PHSC 208 Topic 25.5 2/19/2025  DNA Recombination §Recombination of DNA is the rearrangement of DNA sequences by exchanging segments from different molecules §Exchange of dsDNA between maternal and paternal chromosomes prior to gamete formation causes linkage disequilibrium in genetics. §Two main types of recombination: §General recombination occurs between homologous DNA molecules (most common during meiosis) §Homologous recombination §Occurs in all living organisms §Site-specific recombination—the exchange of sequences only requires short regions of DNA homology §Observed in transposition variation §Observed in bacteriophage DNA integration in E. coli DNA 12 PHSC 208 Topic 25.6 2/19/2025  Holliday Model of General Recombination } Two homologous DNA molecules are paired } Two of the DNA strands are cleaved, one in each } The two nicked strand segments cross over, DNA ligase seals cuts to form a Holliday intermediate } Branch migration, via base- Figure 25.41 pair exchange, leads to transfer of a segment of DNA Figure 25.40 13 PHSC 208 Topic 25.6 2/19/2025  Holliday Model (cont) } Second series of DNA strand cuts occurs on Chi structures } Can be resolved in 2 ways } DNA polymerase fills any gaps, and DNA ligase seals cuts Figure 25.39 Figure 25.40 14 PHSC 208 Topic 25.6 2/19/2025  Proteins Mediate General Recombination } RecBCD } Contains both nuclease and helicase activity } Binds ends of dsDNA and unwinds } Degrades back to specific sites } Chi sequences } Every 5 kB in E. coli } GCTGGTGG } At which it increases rate of 5’ end cleavage } Recruits RecA } Mediate Strand Exchange } ATP dependent } RecA partially unwinds the duplex } Exchanges the ssDNA with the corresponding strand on the dsDNA } 3 stranded intermediate 15 PHSC 208 Topic 25.6 2/19/2025  Holiday Junction Branch Migration and Resolution } RuvA } 2 homotetramers form around Holiday junction } RuvB } ATPase } 2 hexamers form around dsDNA on opposite sides } DNA is pulled through the RuvB rings and pushed apart within RuvA } RuvC } Nuclease that resolves junctions Figure 25-48 16 PHSC 208 Topic 25.6 2/19/2025 Holliday Junction Animations 17 PHSC 208 Topic 25.6 Recombination Repair of Collapsed Replication Forks } Damaged replication forks are commonplace } At least once per bacterial cell division } Around 10x per eukaryotic cell cycle } Thought to be the primary function of homologous recombination } Presence of a nick in the DNA template causes a replication fork collapses } Replisome dissociates } Repair begins with RecBCD and RecA mediating stand invasion of the newly synthesized 3’end into the homologous dsDNA } Branch migration by RuvAB occurs } RuvC resolves } 5’ end of the nick becomes the 5’ end of an Okazaki fragment } Origin-independent replication restart } Restart primosome Figure 25-49 18 PHSC 208 Topic 25.6 2/19/2016  Double Strand Break Repair } Homologous end-joining } Nonmutagenic alternative to double strand break repair to nonhomologous end-joining } Copies sequences from a homologous chromosome } Both dsDNA ends are cut back to yield single-stranded ends } Rad51 mediates strand invasion of a 3’ end } Other 3’ end pairs with the displaces strand } DNA polymerase extends invading and noninvading 3’ ends } Branch migration and Holliday junction resolution } BRCA1 and BRCA2 are proteins that interact with Rad51 } Mutant versions strongly associated with Figure 25.50 cancer 19 PHSC 208 Topic 25.6 2/19/2025  CRISPR-Cas } In addition to restriction endonucleases, prokaryotes contain additional defense mechanisms against viruses } CRISPR } Clustered Regularly Interspersed Short Palindromic Repeats } Arrays of DNA with hundreds of repeating palindromic sequences } 20-50 bp long } Interspersed by unique sequences called protospacers } Protospacers contain DNA sequences from bacteriophage DNA } Transcription of the CRISPR locus generates several ~30 base transcripts } crRNAs } crRNAs bind together with tracRNA to Cas proteins (CRISPR-associated) } CAS proteins have nuclease activity that then recognizes invading DNA complementary to protospacer sequence } Requires particular PAM sequence (protospacer-adjacent Figure 25.51 motif) 20 PHSC 208 Topic 25.6 2/21/2020 CRISPR-Cas for Modifying Genomes } Most common approach to gene knockouts } Can be used to activate specific genes Figure 25.53 21 PHSC 208 Topic 25.6 2/21/2020