Lecture 6

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Mutations and Mutogens

-Mutation: permanent, transmissible change to the genetic material of a cell

-Mutogen: chemical compounds (UV radiation, ionizing radiation) that increase the frequency of mutations

DNA Repair

Proofreading by DNA Polymerase

-DNA pol introduces errors

-E coli introduces errors every 10000 nucs but measured rate is 1 per million

-Some DNA pol have proofreading activity

-In eukaryotes, pol epsilon and delta (not alpha) have 3' to 5' exonucleases or proofreading activity

-Pol detects the error (shift in bp) and shifts from pol -> exonuclease domain and removes 10-20 nucs where the error is

-All prokaryotes have exonuclease fcn

Base Excision Repair (BER)

-Deamination of cytosine forming uracil occurs spontaneously (C->U)

-Cytosine can also be methylated by DNA methyltransferase and gets deaminated and turned into thyamine

-Important for gene regulation, transposons, silencing, chromatin remodeling

-This means that the bp would now be TG instead of CG which doesn't work

-Deamination can therefore lead to point mutation or can result in daughter DNA having mutant TA while wild type has GC

-Steps to fix:

DNA glycosylase hydrolyzes the bond between the mispaired base and the sugar phosphate backbone

The wrong base T is released

Apurinic/apyrimidinic endonuclease 1 (APE 1) cuts the DNA backbone (sugar phosphate)

AP lyase associated with DNA pol beta removes deoxyribose phosphate (the rest of the complex that was bonded to the base T, after the T leaves)

DNA pol beta fills the gap and DNA ligase seals the nick in the backbone

Mismatch Excision Repair

-Error during replication that was missed by DNA pol

-Steps:

MSH2 and MSH6 bind to the daughter strand in such a way to distinguish between the template nuc and the new, erroneous nuc (if gets the wrong one, a point mutation will occur)

Triggers the binding of MLH1 and PMS2 (dimerized)

This complex binds to the endonuclease that cuts the new daughter strand

DNA helicase unwinds and an exonuclease removes several nucs from the cut including the error

Gap is filled by DNA pol delta and DNA ligase

Nucleotide Excision Repair

-Mutagen: UV radiation, chemical adducts

-Effect: modified bases that distort the normal shape of DNA

-Use NER to fix

-Usually the distortion is caused by a thyamine, thyamine covalent bond called a dimer OR addition of aflatoxin B to DNA

-Steps

Dimer recognized by a complex of XP-C and 23 B proteins

Complex recruits the transcription factor TFIIH which unwinds DNA

TFIIH and RPA keep the bubble open with XP-G protein

XP-G now acts as an endonuclease and XPF endonuc cut the strand and remove a section to release the error

Pol fills the gap and ligase seals it

For the next two systems:

-Mutogen is ionizing radiation (X ray), anticancer drugs like bleomycin

-Effect is single or double strand break leading to genomic rearrangements or translocations

-Repair = end end joining or homologuous recombo

Double Strand Break Repair by End Joining

-Can INTRODUCE mutations

-Steps:

Complex formed by DNA-PK (dependant protein kinase) and Ku80/Ku70 heterodimer binds to the end of the break

Ends digested by endonucleases

Ends ligated together

-The break was fixed but because of the digestion of the ends, lost some bp and possibly some proteins

Double Stranded Break Repair by Homologous Recombo

-Can exchange the segments of two double-stranded DNA mol

-Steps:

Broken ends are digested by 5’-exonucleases leaving a single stranded region of DNA with a 3’ end

RecA (prok) or Rad51 (euk) catalyzes the invasion of one of the 3’ ends into the homologous region of the complimentary chromosome

3’ end of the invading strand is extended by DNA pol, displacing the parent strand as an enlarging single strand loop of DNA

When the loop is pushed until it reaches the other end of the cut DNA the complimentary sequences bp

The cut 3’ end extends with the pushed bubbling strand as template DNA

New 3’ ends are ligated (cut) in 2 ways called Holliday structures

Creates two recombinant chromosomes containing DNA of 1 parent mol on one side of the break and DNA of other parent mol on the other side