DNA and the Cell Cycle › DNA Repair

Mismatch Repair

Notes

Mismatch Repair

Sections

DNA REPLICATION: FACTORS THAT INCREASE FIDELITY
• Increase fidelity to 1 mistake per 10^9 bases added

  1. DNA Pol. proofreading capacity
    • DNA Pol. 3' to 5' exonuclease activity: can excise/replace incorrect nucleotides
    • Prokaryotes: DNA Pol. I (replaces RNA primers) & III (daughter strands) are exonucleases
    • Eukaryotes: DNA Pol. delta (lagging strand) & epsilon (leading strand) are exonucleases
  2. Repair mechanisms

Mismatch repair: identifies/fixes replication errors that escape DNA Pol.

• Addresses single-strand breaks in newly replicated DNA
• Cannot repair DNA Damage

MISMATCH REPAIR: E. COLI
• Mismatched nucleotides cannot H-bond --> distorts DNA
• GATC sequence occurs about every thousand nucleotides
• A in GATC is methylated: distinguishes parent strand from daughter strand
• Mut proteins (mismatch repair enzymes in E. Coli)

  1. MutS recognizes mismatched base and initiates repair by binding
  2. MutS forms complex with MutL
  3. MutL binding activates endonuclease MutH
  4. MutH cleaves daughter strand opposite to adenine-methylation (GATC)
  5. Exonuclease and helicase excise portion of daughter strand

Intertextual variation exits regarding above step.

  1. DNA Pol. (III or I) fills gap and ligase seals ends w/ phosphodiester bonds
    • DNA Pol synthesizes gap in 5' to 3' direction

MISMATCH REPAIR: HUMANS
• MSH proteins: human homologs for Mut proteins (no MutH homolog)
• Daughter strand specificity poorly understood: not adenine-methylation

Theoretical recognition sites: breaks in lagging strand & lengthening 3' end of lagging strand.

• DNA Pol. (delta and epsilon) fill excised gap

CLINICAL CORRELATION

Lynch Syndrome

• Formerly known as hereditary nonpolyposis colorectal cancer (HNPCC)
• Mutation in human Mut homologs: defective mismatch repair pathway
• Increased risk for colorectal and other cancers
• ~ 3 in every 100 cases of colon cancer caused by Lynch syndrome

Full-Length Text

  • Here we will learn about a DNA excision repair system called mismatch repair, which corrects mistakes made during DNA replication.
  • To begin, start a table to learn some key points about DNA replication.
  • Denote that DNA has a high fidelity of replication, which means that the frequency of replication errors is very low.
  • Denote that the following increase fidelity:
    • DNA polymerase's proofreading capacity
    • Repair mechanisms.
  • Denote that DNA polymerase has 3 prime to 5 prime exonuclease activity, meaning it can excise an incorrect nucleotide and replace it with the correct one during replication.
  • Denote that the mismatch repair pathway identifies and fixes the replication errors that escape DNA polymerase exonuclease activity.
  • Denote that it only addresses single-strand breaks in newly replicated DNA.
    • It cannot repair DNA damage.
  • Finally denote that these proofreading and repair mechanisms increase the fidelity of DNA replication to only one mistake per 10^9 bases added!

Now, let's illustrate DNA polymerase's proofreading ability.

  • To begin, draw a segment of newly replicated DNA and distinguish the parent and daughter strands.
  • Label the 5 prime and 3 prime ends of each strand.
  • Next, label the following nucleotides on the parent strand: AGAT.
  • Now, on the daughter strand, draw the following nucleotides: TCTC.
  • Circle the last nucleotide on the daughter strand to indicate that it is a mismatch.
    • Thymine pairs with adenine, not cytosine.
  • Next, draw DNA polymerase and show that it excises the mismatched base, cytosine, in the three prime to five prime direction.
    • This is the opposite direction of DNA replication, which proceeds in the five prime to three prime direction.
  • Write that once is excises the incorrect base, it can continue synthesizing DNA in the 5 prime to 3 prime direction.
  • Write that in prokaryotes, DNA polymerase I and DNA polymerase III both have 3 prime to 5 prime exonuclease activity and can both fill the resulting gap. DNA polymerase III normally elongates daughter strands during prokaryotic replication.
    • DNA polymerase I excises RNA primers and replaces them with DNA.
  • Write that in eukaryotes, DNA polymerase delta and epsilon can both excise mismatched nucleotides and fill the gap.
    • Delta on the lagging strand and epsilon on the leading strand.

However, mistakes can still escape DNA polymerase, which brings us to mismatch repair.

We will use prokaryotes, specifically E. Coli, as our model and note some known differences in the eukaryotic pathway. We do this because aspects of the eukaryotic pathway are still poorly understood.

  • Now, draw a DNA daughter strand with a bend in the middle.
    • This bend is our mismatched nucleotide.
  • Label the mismatched nucleotide thymine and the five prime and 3 prime ends of this strand.
  • Draw cytosine above thymine to emphasize that it is incorrectly paired.
  • Now, draw the complementary parent strand with a bend at cytosine.
  • Write that these incorrectly paired nucleotides do not hydrogen bond.
  • Next add the following sequence to the parent strand: G-A-T-C; it occurs about every thousand nucleotides in E. Coli.
  • Draw it again on the other side of cytosine.
  • Next, draw methyl groups attached to the adenines in each of these sequences.
  • Write that this methylation allows mismatch repair enzymes in E. Coli to distinguish the daughter strand from the parent strand.
  • Write that the newly synthesized daughter strand is not yet methylated.

Now, let's illustrate how the mismatch repair enzymes in E. Coli (Mut proteins) repair this daughter strand.

  • Step 1: Redraw our double-stranded DNA with MutS bound to the mismatched nucleotide – MutS protein recognizes the mismatched base and initiates repair by binding it.
  • Step 2: Add MutL to our diagram – MutS forms a complex with MutL.
  • Step 3: Add MutH to our diagram – MutL binding activates the endonuclease MutH.
  • Step 4: show that MutH cleaves the daughter strand opposite to the site of adenine-residue methylation.
  • For step 5: use an arrow to show that an exonuclease and a helicase excise a portion of the daughter strand.
    • Intertextual variation exists regarding this step. Some imply that Mut S, Mut L, an exonuclease and a helicase together excise this portion.
  • Now draw the product of this step: remove the portion of the daughter strand between the MutH nick and the mutation.
  • Step 6: DNA polymerase (either III or I) fills the gap and ligase seals the ends with phosphodiester bonds.
    • Intertextual variation exists regarding which DNA polymerase (III or I) typically fills this gap.
  • Show that DNA polymerase synthesizes the gap in the 5 prime to 3 prime direction.

Finally, let's note the key differences in eukaryotes, specifically in humans.

  • Write that human homologs for Mut proteins are MSH proteins.
  • Specifically write that there is no MutH homolog in humans.
  • Next, write that daughter strand specificity is not dictated by DNA methylation, and is poorly understood in humans. - Some theories suggest that breaks in the lagging strand and the lengthening 3 prime end of the leading strand are recognition points for MSH proteins.
  • Finally, indicate that DNA polymerases delta and epsilon fill the excised gaps in mismatch repair.
  • As a clinical correlation, write that Lynch Syndrome, formerly known as hereditary nonpolyposis colorectal cancer (HNPCC), results from a mutation in human Mut homologs.
    • Patients with Lynch syndrome have a defective mismatch repair pathway and a significantly increased risk for developing colorectal and other cancers.
    • About 3 in every 100 cases of colon cancer are caused by Lynch syndrome.