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• Chapter 25 discusses DNA replication, repair, and recombination
• DNA damage, repair mechanisms, recombination types, and CRISPR-Cas systems are key topics

DNA Damage

• The typical mutation rate is 1.0 mutation per 100,000 genes per generation in germline cells and higher eukaryotes
• Somatic cell mutations are less problematic but can cause cancer
• Environmental and chemical agents can damage DNA to generate mutations
• UV light and ionizing radiation can damage DNA
• Ionizing radiation can generate hydroxyl radicals
• Reactive chemicals and spontaneous purine hydrolysis can damage DNA
• Roughly 20,000 spontaneous purine hydrolyses occur per day out of 6 billion

Types of DNA mutations

• DNA serves as information storage but is not a static molecule
• Single Nucleotide Polymorphisms (SNPs) are single base changes
  • Transitions are when a base is replaced with the same type of base (purine or pyrimidine)
  • Transversions are when a purine is replaced with a pyrimidine, or vice-versa
• SNPs in the protein coding sequence can cause no change, premature stop, or altered protein sequence
• Indels are insertions or deletions, generally involving more than one base (1-1000's)
• Frameshift mutations are indels in the protein coding sequence that aren't a multiple of 3

Chemical Damage Alters Bases

• Nitrous acid can oxidatively deaminate bases
• Reactive oxygen species are part of regular cell metabolism
• Superoxide radicals, radicals of hydroxide, and peroxide can damage DNA
• 8-oxoguanine is an example
• Alkylation of purines at the N7 position promotes hydrolysis of the glycosidic bond, and the repair system is prone to error
• Spontaneous hydrolysis of purines occurs 20,000 times a day out of 6 billion

Ames Test for Mutagenesis

• Most mutagens also act like carcinogens
• Carcinogenesis animal tests can cost a lot and take over 3 years
• The Ames test measures mutagenesis
• Salmonella typhimurium is in histidine
  • Cannot grow unless media is supplemented with histidine
  • May lead to a correct mutation and result in his+
  • 10^9 bacteria are plated and allowed 2 days to wait

DNA Repair Mechanisms

• A typical mammalian cell contains 100,000 DNA molecular lesions/day
• There are a variety of repair mechanisms in cells
• The enzyme photolyase excites electrons to catalyze the retro-Diels-Alder reaction
• Base flipping separates adjacent linked pyrimidine rings
• Placental mammals do not have photolyase
• 06-alkylguanine-DNA alkyltransferase removes a methyl group with a Cys residue, and permanent enzyme methylation occurs

Base Excision Repair

• The N-glycoside linkage of a damaged base is cleaved along with the deoxyribose
• 8-oxoguanine and uracil are in DNA
• Uracil-DNA glycosylase (UDG) identifies and excises U-G mismatches
• Apurinic or apyrimidinic sites are resolved when nucleases remove the residue, and DNA polymerase (pol I in bacteria; DNA polymerase b in mammals) and DNA ligase fill the gap

Nucleotide Excision Repair

• Exists in all cells
• Corrects pyrimidine dimers and bulky lesions which cause distortion to bases
• The UvrABC endonuclease system works in E. coli
• In the damaged DNA strand, two cuts are made one on each side of the damage
  • E. coli excision nucleases: Uvr A, B, and C
• UvrD helicase removes 11-12 excised DNA bases
• The gap closes, and the nick is sealed
• Xeroderma Pigmentosum: disorder marked by deficiency in repair activities and sensitivity to light

Mismatch Repair

• Single strand repair mechanism that restores helix distorting mispairings
• Proofreading and replication errors which escaped editing functions during transcription get fixed
• Distinguishes between new and old synthesized strands
• In prokaryotes, methylation tags the template strand, and MutS homodimer recognizes mismatches
• Proteins are recruited to selectively cleave the portion of the new strand
• Homologs of Muts and L are in eukaryotes, often using lagging strand status to tag the new strand

Double Strand Break Repair

• Inter strand crosslinks, topoisomerase inhibition/deficiency, and ionizing radiation damage cause double strand breaks
• 5-10% break of dividing cells in culture show a chromosomal break
• There are two repair pathways
  • By removing or extending ssDNA and bringing ligation of two ends brings repair
  • End-joining core complex: Ku protein
• Subject to error due to no sequence homology needed

DNA Recombination

• DNA recombination rearranges DNA seuqences by exchanging segments from molecules
• Transfer of dsDNA from maternal and paternal chromosomes during gamete formation causes linkage disequilibrium in genetics
• Occurs between homologous DNA molecules (most common during meiosis)
  • Homologous recombination is in living organisms
• exchange requires short DNA homology regions
  • Observed in transposition variation
  • Observed in bacteriophage DNA integration in E. coli DNA

Holliday Model of General Recombination

• Two homologous DNA molecules are paired

• The two DNA strands get cleaved, one in each

• Nicked strand segments DNA ligase seals cuts to form a

• Transfer leads of a DNA segment due to base pairing via branch migration

• After a series of DNA strand, cuts the event may occur on

• May resolve in two ways

• DNA Pol fills in any gaps, then DNA ligase seals the cuts

Proteins Mediate General Recombination

• RecBCD:
  • Contains nuclease and helicase activity
  • Binds and unwinds the ends of dsDNA
  • Degrades back to specific Chi sites in E. coli every ~5 kb
• Recruits RecA:
  • It increases rate of 5' end cleavage and mediates Strand Exchange
  • It's ATP dependent
  • RecA only partially unwinds double stranded DNA
  • Exchanges ssDNA with the corresponding strand on the dsDNA, and resulting in 3 stranded intermediate

Holiday Junction Branch Migration and Resolution

• RuvA: Two homotetramers are formed around the Holiday junction
• RuvB:
  • ATPase
  • Forms around double stranded DNA on opposite sides
  • RuvB rings pull the DNA through; RuvA pushes them apart within it
• RuvC: Nuclease that helps resolve junctions.

###Recombination Repair of Collapsed Replication Forks

• Commonplace damaged replication forks occurring
  • At least once per bacterial cell division
  • Eukaryotic cell cycle around 10x
    • Thought to be homologous recombination
• In the DNA template, a nick causes collapse of replication fork
  • Replisome dissociates
• Fixed by first using RecBCD and RecA to mediate invasion of the newly created 3' end in homologous dsDNA
• Branch Migration by RuvAB occurs, and gets resolved via RuvC
• The 5' end of the nick then becomes the 5' end of an Okazaki fragment
  • Origin-independent replication restart
    • Restart includes primosome

Double Strand Break Repair Overview

• Homologous End-Joining
  • Serves instead nonmutagenic, to double-stranded breaks
  • Duplicates sequences from homologous chromosomes and both dsDNA ends are cut back to yield single-stranded ends
  • Rad51 mediates strand invasion on 3' end, and the pairs with the displacing strand end
  • Non-invading and invading 3' ends get extended by DNA polymerase
  • Branch migration and Holliday junction resolution occur
• BRCA1 and BRCA2 are proteins that work with Rad51
  • Cancer comes from mutant versions

CRISPR-Cas System

• Prokarytoes contain defense mechanisms against more than just restriction

  • CRISPR is a defense mechanism against viruses
  • Arrays of DNA contain repeating palindromic sequences and short palindromic repeats
    • The repeating regions of the sequences are referred to as CRISPR
    • 20-50 base pairs long
    • Unique regions that separate it are called protospacers
  • Protospacers contain DNA sequences from bacteriophage DNA
  • Several ~30 base transcripts comes the CRISPR locus transcription
  • crRNAs binds trRNA to Cas (CRISPR associated) proteins
  • Recognizes DNA complementary to its protospacer sequence with nuclease
  • Necessitates specific PAM sequence (protospacer-adjacent motive)

• Some approaches to modifying genomes using CRISPR/Cas include:

  • Common goal: gene knockouts
  • Activating specific genes