Lecture 7 DNA repairChapter 10

Problems 2, 4, 6, 8, 10, 12, and 14

Quiz 3 due today at 4:00 PM

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Types of mutations

Transition mutations Transversion mutations

Pyrimidine to __________Purine to ___________

Pyrimidine to __________Purine to ___________

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Mutations can be permanently fixed if they are not repairedbefore the next round of replication

Polymerase errors cause a distortion of the DNA helix

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Mismatch repair of mutations in E. coli

MutS protein recognizes mismatch, induces a kink in the DNA, and binds ATP

MutS recruits MutL and MutH

MutL activates MutH. MutH nicks DNA

Helicase unwinds strand. An exonuclease degrades the strand with the mutation

Polymerase III fills in the gap

10_Figure04.jpgMutS complexed with DNA

kink in DNA

ATP

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How does the cell knowwhich strand contains the mutated base?

Dam methylase does notmethylate right after replication. Later A in GATC sequencesare methylated.

MutH nicks the unmethylatedstrand of DNA in the mismatchrepair process

10_Figure06.jpg Directionality in mismatch repair

The exonuclease binds the MutH nick and degrades DNA travelling toward MutS until MutS and the mismatch are found

Exonucleases have a distinct polarity (5’->3’ or 3’->5’) so at least2 different exonucleases must be used in mismatch repair

Eukaryotic cells lack a MutH homolog

Then how do the mismatch repair enzymes recognize the newly synthesized strand of DNA?

Nicks present in unligated Okazaki fragments may play this role following lagging strand DNA

synthesis.

But how nicks get introduced in the leading strand DNA is not quite clear.

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Hydrolytic damage of DNA

U base pairs with AWhat is the result?

Adenine and guanine also spontaneously deaminate tohypoxanthine and xanthine.

Which type of damage is worst?

Why didn’t DNA evolve to use U?

deamination

depurination

deamination

Abasic site (apurinic deoxyribose)

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Ames Test to detect mutagens (carcinogens)

A base substitution or frameshift mutation is introduced in a gene used to make the amino acid histidine.

Some mutagens needto be activated in the liver.So liver extract is commonlyadded to the mutagen.

Detects reversionsof mutation

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Base damage by alkylation and oxidation

Alkylation – introduction of methyl or ethyl groups by chemicals (nitrosamines)

Can form O6-methylguaninewhen alkylated

8-oxoguanine (Oxo-G) forms after oxidation. It can basepair with A.Would this lead to transition or transversion mutation?

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Radiation-induced DNA damageUV light causes adjacent pyrimidines to covalently bond.This blocks progression of DNA polymerase.

Gamma and X-ray radiation and some drugs (bleomycin)induce double stranded breaks in DNA

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Chemicals that cause mutations in DNA

Base analogof thymidine

What is result?

Intercalating agents-insert between basescausing insertions& deletions in DNA

What type ofinteraction withbases?

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DNA repair systems10

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Direct DNA repair: Photoreactivation

Visible light is used as the energy to repair thymine dimers

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Direct DNA repair: methyl group removal

Repair of alkylation of O6-methylguanine

A cysteine on the methyltransferase binds the methylgroup on guanine.

10_Figure13.jpg Base excision repair pathwayExample: uracil glycosylase

What type of site is present after glycosylase action?8 different DNA glycosylases in humans (ex. Oxo-G).They scan minor groove looking for damaged basesand then use base flipping to access base for repair.

How does uracil usually get in DNA?

AP site

10_Figure14.jpg Oxo-G glycosylase

DNA is purple

Glycosylase protein is gray.

What is differentabout the red base?

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Oxo-G:A repair

It’s not always too late to repair DNA after replication.A glycosylase recognizes oxo-G:A and removes the A.

Another glycosylase recognizes G:T basepairs and removes the T which likely arose from spontaneousdeamination of 5-methylcytosine.

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Nucleotide excision repair in E. coli

Nucleotide excision repair recognizesdistortions in the double helix.

UvrA+UvrB scan DNA.UvrA recognizes distortion and leaves.UvrB melts DNA to form single-stranded bubble

UvrC is recruited and cuts DNA 8 nucleotides5’ of the legion and 4-5 nucleotides 3’ ofthe legion.

Helicase UvrD removes the single strand.DNA polymerase I and DNA ligase fill the gap.

Similar but more complex than NER in E. Coli.

Mammalian NER uses around 25 proteins.

XPC recognizes distortions (like UvrA in E. coli).

XPA and XPD helicases melt DNA (like UvrB in E. coli).

Single stranded binding protein RPA binds DNA.

5’-cleavage site cut by ERCC1-XPF nuclease and 3’-cleavage site cut by XPG nuclease (similar to UvrC in E. coli)

24-32 nucleotide long DNA strand is released that is filled in

by a polymerase and sealed by DNA ligase.

Xeroderma pigmentosum disease caused by mutations in XP_ (NER) genes. Patients are susceptiple to cancer from UV light.

Nucleotide excision repair (NER) in humans

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Transcription coupled DNA repair

In humans when transcription of DNA stalls due to a lesion in DNA, the RNA polymerase recruits the NER proteins.

The TFIIH complex needed for melting DNA for transcription contains XPA andXPD. What is the significance of this?

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Mammalian non-homologous end joining(NHEJ) pathway to repair double strandedDNA breaks.

NHEJ also used in VDJ recombinationto produce staggering amounts ofdifferent types of antibodies to fight infections and in Bacillus subtilisbacterial spores to protect the DNA.

Double stranded breaks (DSB) arethe most toxic of all types of DNA damage.

Ku70/Ku80 heterodimer binds ends of DNA and recruits DNA-protein kinase cs.Artemis, an exo/endonuclease, is phosphorylated by DNA-PKcs andprocesses the DNA ends. Ligase IVcomplex attaches the 2 ends together.

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Translesion DNA synthesis in E. Coli

Erroneous and used as last resort, but used to replicate through DNA lesions. Sliding clamp and DNA Pol III fall off DNA.

A translesion polymerase (Pol IV or Pol V)copies across lesion (thymidine dimer).

Translesion polymerase falls off and DNA Pol III holocomplex resumes replication.

UmuC part of Y family of DNA polymerases.

Pol V contains UmuC

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Y family polymerase (left) & high fidelity T7 phage polymerase (right)

translesionpolymerase

Incoming nucleotides in red and template in blueWhat do you notice about the structure around the active site (yellow arrow)?

normal polymerase

10_UnFigure02.jpg The Y Family of translesion polymerases

Translesion DNA synthesis in E. Coli is induced by SOS DNA damage response.Adding ubiquitin peptide to sliding clamp at lesion recruits translesion polymerase.

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DNA repair systems10

Double strand break repair pathway(homologous recombination)

Uses information from homologous sister chromosome and will be discussed next

lecture (chapter 11)