Error Repair Mechanisms

• Introduction
  • repair is obviously a vital process to any organism, in terms of maintaining the integrity of the hereditary "program"
  • recently, the subject has been getting even more attention, because several of the most highly publicized "cancer genes" turn out to be mutations in error repair processes in humans.
• Types of error repair
  • we already covered proofreading by DNA polymerase
  • methyl-directed mismatch repair is also very closely associated with replication
    • fixes mismatches that get past the proofreader
      • imagine that PolIII misincorporates a G across from a T during replication
      • perhaps it's a tautomeric shift that can't be proofed by epsilon
    • now the problem is knowing which strand is correct and which is has the error
      • rationale is something like this: the cell marks DNA "old", and then assumes that old DNA is correct
      • so in a mismatch, the error is always assumed to by "new" DNA.
    • the molecular event that marks DNA as "old" (and therefore, completely proofread and free of errors), is methylation of particular bases in DNA.
      • the dam methylase (dam stands for deficient in adenine methylation) recognizes the palindrome GATC and places a methyl group on the A
      • this occurs after replication, but there is a slight lag in time between replication and methylation
      • due to the lag, for a short time, DNA is hemimethylated
        • the template strand has a methylated A from the previous round of replication
        • complementary strand that has just been synthesized does not have any methylation
      • so..., as long as the cell can sense the methylation state of DNA, it can distinguish the template strand from the newly synthesized strand.
    • the mismatch repair system features a protein (MutH) that recognizes and binds to hemimethylated DNA
    • in addition two proteins (MutS and MutL) bind to the mismatch.
    • the combination of MutH bound at GATC and MutSL at mismatch causes MutH to nick DNA on the unmethylated strand only.
    • a nick provides a 3' OH group, which acts something like a primer for DNA polymerase
      • the nick triggers the nick translation activity of DNA polymerase, which plows right through the mismatch and repairs it
      • then, just as in replication, ligase must seal the nick
    • finally, the dam methylase methylates the new strand, and the DNA is assumed to be completely proofread.
    • and by the way, mutations in the human homologues of mutS and mutL cause individuals to have a much greater chance of having a type of cancer called Hereditary Nonpolyposis Colon Cancer (HNPCC)
  • photorepair is the easiest to understand
    • it is one of the very few mechanisms that fix damage without removing bases or nucleotides
    • fixes they cyclobutane pyrimidine dimers that are caused by absorption of UV light by DNA
    • the enzyme photolyase simply binds to the dimers (which are on the same strand) and breaks the cyclobutane ring.
    • it's called photorepair because photolyase only works in the light
    • humans don't have this enzyme.
  • excision repair fixes all kinds of damage to DNA
    • best characterized type is the uvrABC endonuclease ("short patch")
      • UvrAB scans the DNA for distortions in double helix
      • if it finds one, it recruits UvrC, and UvrA leaves the DNA
      • UvrBC nick the DNA on the damaged strand, both upstream and downstream of the damage
      • UvrD is a helicase that simply removes the piece of DNA with the damage
      • DNA polymerase  and ligase seal in the "patch", using the remaining undamaged strand as template.
      • other excision repair systems only remove the damaged or mismatched nucleotide ("very short patch")
  • recombinational repair of pyrimidine dimers
    • during replication, polIII can't handle a dimer, so it just gives up, and skips down the DNA a bit, leaving a single stranded region with no new DNA.
    • RecA can recognize the ssDNA, and cause it to synapse with the other double strand
    • then, DNA is swapped, so that the region complementary to the dimer is correct, and polI can fill in the gap left in the other double helix
    • the bottom line is the dimer doesn't get fixed by recombinational repair.  It just gets diluted out (and perhaps fixed later on).
  • SOS response

  
   

• Homologous Recombination

  • significance of recombination
    • process probably occurs in all living things
    • allows the organism to "shuffle the genetic deck" so to speak, perhaps producing new combinations of genes that will work better in a particular environment
    • in eukaryotes, it helps insure that siblings are not genetically alike
    • in prokaryotes, it provides a way for organisms to move genes from chromosome to plasmid and back
    • in general, it speeds up the process of evolution
    • ...and, it is involved in error repair
  • molecular basis of homologous recombination
    • first of all, remember that for HOMOLOGOUS recombination, the DNA sequences that recombine must be the same or very similar
    • overview: the Holliday model
    • 
      • one strand of each DNA molecule is nicked at the same positions
      • the two molecules pair and are ligated to form a Holliday junction
      • the junction can be isomerized to form two structures
      • "resolution" (i.e., cutting and ligating) produces a patch or exchange, depending on the isomer that is resolved.
    • the role of RecBCD (also known as exonuclease V) and Chi sites
      • chi sites are eight base sequenses (consensus is 5'GCTGGTGG3') that tell the RecBCD complex what to do
      • here is how it works: RecBCD binds to ends of DNA, then migrates along the DNA, producing single stranded loops that its exonuclease activity degrades.  As soon as RecBCD encounters a chi site, it stops degrading the ssDNA.  This results in the formation of a strand that can synapse and recombine with a homologous strand
    • synapsis requires RecA, a major player in the life of a cell.
      • RecA is a small protein with a big set of functions
        • it's a co-protease involved in global response networks like SOS
        • it's an ATP driven helicase
        • it's a "synapsase" (I made this word up) that figures in the final steps of homologous recombination
      • RecA coats the single stranded DNA that the RecBCD complex made (see above) to produce a helical filament
      • this filament can interact with another double helix to form triple-helical DNA
      • if the strands are similar enough, the incoming strand can displace one of the strands of the original double helix, and recombination can take place.
    • gap repair synthesis