Mutations_09.27

• Cell Cycle Checkpoint activation!
• Needs to repair damage before the cells are reproduced. Can prioritize DNA repair
• 

apoptosis

• DNA Damage Response (DDR)
• 

E.coli

True

through identifying and characterizing mutants defective in DNA repair

• Fixes Damage immediately upon recognizing it
• ex: DNA photolysase can reverse formation of pyrimidine dimers by UV radiation

in E.coli, pyrimidine dimers are induced by UV

Photolyase uses the energy of light: the enzyme contains a chromophore that absorbs light in the UV/blue range (300-500nm wavelength)

This photoreactivation of photolyase results in cleavage of the pyrimidine dimer

• There are four steps in this mechanism:
• 1)Photolyase recognizes and binds to the damage
• 2)Light absorption by chromophore converts it to an excited state
• 3)Chromophore donates an electron to the cyclobutyl dimer
• 4)Dimer is destabilized and undergoes a series of electron rearrangements which result in monomeric pyrimidines

• Photolyase contains two chromophores that abosrb light energy
• 1. FADH-
• 2. either (5,10-MTHF) or (8-HDF)
• The light is gathered by chromophore (2) and energy is transfered to FADH-
• This energy is used to split the dimer

bacteria, fungi, plants, and many vertebrates but not placental mammals

• (5,10-MTHF) methenyltetrahydrofolate
• OR
• (8-HDF) 8-hydroxy-5-deazaflavin

FADH-

Multicellularity. We only currently need it in our skin cells, the other organs, so many of our other cells have no use for the enzyme. An unused enzyme is lost

• Photolyase
• AGT
• AlkB

• A homologous AGT protein present in bacteria, O6-alkyl-guanine repaired by transfering alkyl group to the AGT protein in the basic repair pathway
• When Ada protein is methylated, it becomes a transcription factor that induces expression of its own gene

• AGT transfering a alkyl group from O-6-alklyl guanine to itself
• This transfer repairs the guanine and inactivates the AGT protein

• oxidatevly demethylates the DNA substrate
• present in all living organisms
• reverses methylation on nucleic bases
• hydroxylates the methyl group, to restore the original base

• Most commonly used to repair small base lesions that don't distort the helix
• Abnormal bases are recognized by DNA glycosylase and binds to the damaged DNA to initiate a repair pathway to remove the base

Abnormal bases are recognized by DNA glycosylase and binds to the damaged DNA to initiate a repair pathway to remove the base

• 1. DNA glycosylase binds to damaged base to cleave the nucleotide at the glycosidic bond
• 2. AP endonuclease binds and cleaves DNA upstream of the Damage
• 3. Exonuclease removes the segment of DNA (including AP site on the backbone)
• 4. DNA Pol I fills in the gap
• 5. DNA Ligase seals the nick

• Short-patch BER
• long-patch BER
• 

• DNA glycosylases recognizes and removes the damaged base
• 5’ incision by Apurinic Endonuclease 1 (APE1)
• a dRP lyase removes the deoxyribose phosphate and DNA pol β subsequently fills the gap by incorporating a single nucleotide

DNA ligase III with its accessory protein XRCC1 seals the gap

The long-patch BER also first relies on DNA glycosylases and APE1

Next, DNA pols δ, e (or β) synthesizes 2–11 bases displacing the damaged abasic site-containing DNA strand

PCNA clamp and RFC clamp loader assist the DNA Pol

The displaced ssDNA flap is cut by FEN-1

The nick is sealed by DNA ligase I

DNA pol β

DNA pol δ,ε

• It requires a DNA glycosylase to recognize each specific damaged site
• too many ways to mutate DNA (chemical, radiation, oxygen radicals...)

• NER machinery uses a limited number of proteins to recognize damaged regions in DNA based off of abnormal structure or chemistry.
• Excises and replaces these regions

12 nucleotides

30

True

• False
• we studied bacterial and mammal (short vs long-patch repair)

Damage recognition

Binding of a protein complex at the damaged site

Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides

Removal of the damage-containing fragment from between the two nicks

Filling in of the resulting gap by a DNA polymerase

Ligation

ABC excinuclease

Uses the protein complex ABC excinuclease which consists of many smaller parts mentioned below:


Two UvrA proteins form a complex with one UvrB protein in an ATP-dependent reaction

The complex scans DNA and recognizes UV damage by distortion in the helix

The UvrA proteins dissociate from  the complex after ATP hydrolysis

This leaves UvrB bound across from the damage


Next, UvrB recruits UvrC protein to the complex

UvrC activates UvrB to nick the DNA four nt 3’ from the pyrimidine dimer

Then UvrB activates UvrC to nick the DNA seven nt 5’ from the pyrimidine dimer

This leaves a fragment of DNA containing the damage that can now be removed


Next, UvrD helicase uses ATP hydrolysis to power the unwinding of the damaged DNA fragment (this removes UvrC)

The gap in the DNA is now filled in by DNA Pol I or II, removing UvrB in the process

Finally, DNA ligase seals the nick

The end steps of the NER pathway in e.coli


Next, UvrD helicase uses ATP hydrolysis to power the unwinding of the damaged DNA fragment (this removes UvrC)

The gap in the DNA is now filled in by DNA Pol I or II, removing UvrB in the process

Finally, DNA ligase seals the nick

Middle steps of the NER pathway in E.coli

Next, UvrB recruits UvrC protein to the complex

UvrC activates UvrB to nick the DNA four nt 3’ from the pyrimidine dimer

Then UvrB activates UvrC to nick the DNA seven nt 5’ from the pyrimidine dimer

This leaves a fragment of DNA containing the damage that can now be removed

Two UvrA proteins form a complex with one UvrB protein in an ATP-dependent reaction

The complex scans DNA and recognizes UV damage by distortion in the helix

The UvrA proteins dissociate from  the complex after ATP hydrolysis

This leaves UvrB bound across from the damage

over 30 have been implicated!

Base Excision Repair (BER)