DNA Repair

DNA replication Errors The consequences of DNA replication errors, unless fixed before the next round of cell division, are permanent.

DNA replication Errors

DNA pol I and III proofread 3’ – 5’ direction

DNA Polymerase Editing How can mistakes happen? “Delete Key” 3’ exonuclease (can only cleave DNA from the 3’ end) The mismatched pair alters the geometry between the 3’-OH and incoming nucleotide resulting in poor interactions. However, the mismatched pair is positioned so that the exonuclease region of the DNA polymerase can remove the nucleotide and DNA synthesis can resume.

DNA replication Errors DNA replication errors are commonly repaired by the DNA proofreading mechanism (3’ to 5’ exonuclease function) From wiki

DNA Mismatch Repair What is mismatch repair? A system of recognizing and repairing errors that occur during DNA replication and/or repairing errors caused by DNA damage. There are many different types of mismatch repair systems; however, mismatch repair mechanisms are strand-specific. Why is this important? From wiki

Mismatch Repair in Prokaryotes Main challenges mismatch repair proteins face: Must scan the genome for mismatches (sensed as a distortion of the double helix) 2. Must repair before the next round of replication 3. Must repair the “mutant” nucleotide on the newly synthesized strand!

MutS is an amazingly shaped protein! Peggy Hsieh, Ph.D.

Mismatch Repair in Prokaryotes Mismatch repair systems—removes single base pair errors that escape proofreading Well studied in E. coli Proteins are called the Mut proteins MutS: recognizes mismatches MutH: nicks one strand ahead of the mutation A helicase and exonuclease are recruited to remove base pairs including the mismatch DNA Pol III fills in the gap Sealed with ligase Mut S How does the Mismatch Repair System “know” which strand has the mutation: Can you think of what is different between the parental and newly synthesized strand?

Mismatch Repair in Prokaryotes Mut H only nicks on the unmethylated strand! What enzyme methylates this? DAM: DNA Methyl Transferase Predict what would happen to mutation rates in a Dam(-) bacterial cell

Mismatch Repair in Eukaryotes How would we know if humans have homologs to Mut proteins? MutS Homologs (MSH) were discovered by analyzing for similarities in protein sequences. Mutations in MSH genes result in hereditary nonpolyposis colorectral cancers Would you predict that MSH proteins rely on the same methylation trick? No! Replication in euks does not use the methylation trick! Still ongoing questions regarding this. MSH proteins may recognize the spaces in okazaki fragment and use this an indicator.

Mismatch Repair in Eukaryotes

AP Endonuclease

DNA Glycosylase