DNA Damage and Mutations

DNA Damage

• Mutations are caused by metabolic activities or environmental exposure
• The natural rate of mutation is about 1.0 mutation per 100,000 genes per generation in germline, higher eukaryotes
• Somatic cell mutations are less problematic, but can cause cancer
• Environmental and chemical agents can damage DNA, leading to mutations
• Mutation can be induced via UV light and ionizing radiation
• Hydroxyl radicals can be generated by oxidation of water
• Reactive chemicals can induce DNA damage
• Spontaneous purine hydrolysis occurs at a rate of 20,000 events per day for every 6 billion bases

Types of DNA Mutations

• DNA is designed for information storage but is not a static molecule

• Point mutations involve a single base change and are single nucleotide polymorphisms (SNPs)

• Transitions: A base is replaced with the same type, either pyrimidine (Py) or purine (Pu)

• Transversions: A purine is replaced by a pyrimidine or vice versa

• SNPs can be classified based on their effect on protein coding sequence

  • Silent mutations: No change in protein sequence
  • Nonsense: Premature stop to synthesis
  • Missense: Alters protein sequence

• Indels are insertions or deletions

  • They generally involve more than one base (1-1000’s)
  • Frameshift mutations are indels in protein coding sequence with the number of inserted or deleted bases not a multiple of 3

Altered Bases and Chemical Damage

• Nitrous acid can oxidatively deaminate bases
  • It can cause both AT to GC and GC to AT transitions
• Reactive oxygen species are part of normal cell metabolism
  • Superoxide, hydroxy radicals, and peroxide
  • 8-oxoguanine can yield a GC to TA transversion
• Alkylation of purines at the N7 position promotes hydrolysis of the glycosidic bond
  • The repair system for alkylation is error prone
• About 20,000 spontaneous hydrolysis events of purines occurs per day out of 6 billion per day

Ames Test

• Many mutagens are also carcinogens
• Standard animal tests for carcinogenesis are expansive and can take up to 3 years
• The Ames test is a simple test for mutagenesis
  • Uses Salmonella typhimurium bacteria
  • The bacteria cannot grow unless the media is supplemented with histidine
  • If a correct mutation arises, the bacteria can grow without histidine, becoming his+
  • 10^9 bacteria are plated and observed for 2 days

DNA Repair

• A typical mammalian cell encounters approximately 100,000 molecular lesions to its DNA per day
• multiple repair mechanisms are required
• Direct Reversal of Damage
  • Photolyases use light energy to excite electrons in the ring catalyzing the retro-Diels-Alder reaction
  • Separates adjacent linked pyrimidine rings by base flipping
  • Mammals do not have photolyase
• O6-alkylguanine-DNA alkyltransferase
  • Removes methyl group with a Cys residue from guanine, repairing alkylation damage
  • Enzyme has permanent methylation and transfers alkyl group away forever

Base Excision Repair

• DNA glycosylases cleave the N-glycosidic linkage of a damaged base and the deoxyribose, i.e. the backbone
• Uracil-DNA glycosylase (UDG) recognizes U-G mismatch and excises uracil
• apurinic/apyrimidinic (AP) sites are resolved through the action of nucleases that remove the residue
  • DNA polymerase (pol I in bacteria and polymerase b in mammals) and DNA ligase resynthesize and seals
• BER removes damaged or modified bases, and a base is excised and then replaced

Nucleotide Excision Repair

• Contained by all cells
• Corrects pyrimidine dimers and other bulky lesions that distort the bases from their normal positions, using a UvrABC endonuclease system in E. coli
• Two cuts are made in the DNA in the damaged strand, one from either side of the damage
  • E. coli excision nucleases Uvr A, B, and C perform the cuts
• Excised DNA of ~11-12 bases is removed by UvrD helicase
• The gap is filled, and the nick is ligated
• Xeroderma Pigmentosum (XP)
  • A disorder where patients are deficient in activities needed for this kind of repair, leading to them being extremely sensitive to light

Mismatch Repair

• A single-strand repair mechanism that corrects helix-distorting base mispairings and mismatched based during RNA during genetic recombination
• Mutations in proofreading errors, replication slippage, and transcription
• Relies on its capacity to distinguish between old and newly synthesized strands
• In prokaryotes, methylation marks template strand
  • MutS homodimer recognizes mismatches
  • Additional proteins are recruited to selectively cleave out a portion of the new strand
• Eukaryotes have homologs of MutS and L and may use lagging strand status to mark the new strand instead of methylation

Double-Strand Break Repair

• Double-strand breaks occur with inter-strand cross-links, topoisomerase inhibition/deficiency, and with ionizing radiation damage
• 5% to 10% of dividing cells in culture exhibit a chromosomal break
• Two pathways
  • Non-homologous end joining (NHEJ)
    • removes or extends ssDNA and brings two ends together for ligation
    • the core of end-joining complex is the Ku protein
    • is error prone
  • Homologous Recombination

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, and occurs in all living organisms
  • Site-specific recombination – the exchange of sequences only requires short regions of DNA homology
    • Observed in transposition variation and in bacteriophage DNA integration in E. coli DNA

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 and DNA ligase seals cuts to form a Holliday intermediate
• Branch migration, via base-pair exchange, leads to transfer of a segment of DNA
• Second series of DNA strand cuts occurs on Chi structures
• Can be resolved in two ways
• DNA polymerase fills in any gaps, and DNA ligase seals cuts

Proteins that Mediate General Recombination

• RecBCD
  • Contains both nuclease and helicase activity
  • Binds ends of dsDNA and unwinds
  • Degrades back to specific sites
  • Recognizes Chi sequences in E. coli like GCTGGTGG at which it increases the rate of 5’ end cleavage
• Recruits RecA to mediate Strand Exchange
  • Requires ATP
  • RecA partially unwinds the duplex and exchanges the ssDNA with the corresponding strand on the dsDNA
  • Forms a 3 stranded intermediate
• RuvA
  • 2 homotetramers form around the Holliday 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 the Holliday junctions

Recombination Repair of Collapsed Replication Forks

• Damaged replication forks are commonplace
  • At least once per bacterial cell division and around 10x in eukaryotes
• Thought to be the primary function of homologous recombination
• Presence of a nick in the DNA template causes a replication fork to collapse
  • Replisomes dissociate
• 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 the Holliday junction
• A 5' end of the nick becomes the 5' end of an Okazaki fragment
• Origin-independent replication restart leads to restarting the primosome

Double Strand Break Repair

• Homologous end-joining
  • Nonmutagenic alternative to double strand break repair involving 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 displaced 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 cancer

CRISPR-Cas

• In addition to restriction endonucleases, prokaryotes contain additional defense mechanisms against viruses
• Clustered Regularly Interspersed Short Palindromic Repeats arrays of DNA with hundreds of repeating palindromic sequences that are 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, creating crRNAs
• crRNAs bind with tracRNA to create a Cas-associated complex
• Cas proteins have nuclease activity that recognizes invading DNA complementary to the protospacer sequence
• Requires particular PAM sequence (protospacer-adjacent motif)

CRISPR-Cas for Modifying Genomes

• Most common approach is to generate gene knockouts
• Can be used to activate specific genes