Lecture 7 DNA repair Chapter 10 Problems 2, 4, 6, 8, 10, 12, and 14 Molecular Biology of the Gene Lecture 7 2/6/2013 Lecture 7 DNA repair Chapter 10 Problems 2, 4, 6, 8, 10, 12, and 14 Quiz 3 due today at 4:00 PM

Transversion mutations 10_Figure01.jpg Types of mutations Transition mutations Transversion mutations Pyrimidine to __________ Purine to ___________ Pyrimidine to __________ Purine to ___________

Mutations can be permanently fixed if they are not repaired 10_Figure02.jpg Mutations can be permanently fixed if they are not repaired before the next round of replication Polymerase errors cause a distortion of the DNA helix

Mismatch repair of mutations in E. coli 10_Figure03.jpg 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

MutS complexed with DNA 10_Figure04.jpg MutS complexed with DNA ATP kink in DNA

which strand contains the mutated base? 10_Figure05.jpg How does the cell know which strand contains the mutated base? Dam methylase does not methylate right after replication. Later A in GATC sequences are methylated. MutH nicks the unmethylated strand of DNA in the mismatch repair process

Directionality in mismatch repair 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 least 2 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.

Hydrolytic damage of DNA 10_Figure07.jpg Hydrolytic damage of DNA U base pairs with A What is the result? Adenine and guanine also spontaneously deaminate to hypoxanthine and xanthine. Which type of damage is worst? Why didn’t DNA evolve to use U? deamination Abasic site (apurinic deoxyribose) depurination deamination

Ames Test to detect mutagens (carcinogens) 10_UnFigure01.jpg Ames Test to detect mutagens (carcinogens) A base substitution or frameshift mutation is introduced in a gene used to make the amino acid histidine. Detects reversions of mutation Some mutagens need to be activated in the liver. So liver extract is commonly added to the mutagen.

Base damage by alkylation and oxidation 10_Figure08.jpg Base damage by alkylation and oxidation Alkylation – introduction of methyl or ethyl groups by chemicals (nitrosamines) Can form O6-methylguanine when alkylated 8-oxoguanine (Oxo-G) forms after oxidation. It can basepair with A. Would this lead to transition or transversion mutation?

Radiation-induced DNA damage 10_Figure09.jpg Radiation-induced DNA damage UV 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

Chemicals that cause mutations in DNA 10_Figure10.jpg Chemicals that cause mutations in DNA Base analog of thymidine What is result? Intercalating agents- insert between bases causing insertions & deletions in DNA What type of interaction with bases?

10_Table01.jpg DNA repair systems 10

Direct DNA repair: Photoreactivation 10_Figure11.jpg Direct DNA repair: Photoreactivation Visible light is used as the energy to repair thymine dimers

Direct DNA repair: methyl group removal 10_Figure12.jpg Direct DNA repair: methyl group removal Repair of alkylation of O6-methylguanine A cysteine on the methyltransferase binds the methyl group on guanine.

Base excision repair pathway Example: uracil glycosylase 10_Figure13.jpg Base excision repair pathway Example: uracil glycosylase How does uracil usually get in DNA? AP site 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 bases and then use base flipping to access base for repair.

Glycosylase protein is gray. 10_Figure14.jpg Oxo-G glycosylase Glycosylase protein is gray. What is different about the red base? DNA is purple

It’s not always too late to repair DNA after replication. 10_Figure15.jpg 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 spontaneous deamination of 5-methylcytosine.

Nucleotide excision repair in E. coli 10_Figure16.jpg Nucleotide excision repair in E. coli Nucleotide excision repair recognizes distortions 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 nucleotides 5’ of the legion and 4-5 nucleotides 3’ of the legion. Helicase UvrD removes the single strand. DNA polymerase I and DNA ligase fill the gap.

Nucleotide excision repair (NER) in humans 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.

Transcription coupled DNA repair 10_Figure17.jpg 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 and XPD. What is the significance of this?

Mammalian non-homologous end joining 10_Figure18.jpg Mammalian non-homologous end joining (NHEJ) pathway to repair double stranded DNA breaks. Double stranded breaks (DSB) are the 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 and processes the DNA ends. Ligase IV complex attaches the 2 ends together. NHEJ also used in VDJ recombination to produce staggering amounts of different types of antibodies to fight infections and in Bacillus subtilis bacterial spores to protect the DNA.

Translesion DNA synthesis in E. Coli 10_Figure20.jpg 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

Incoming nucleotides in red and template in blue 10_Figure21.jpg Y family polymerase (left) & high fidelity T7 phage polymerase (right) translesion polymerase normal polymerase Incoming nucleotides in red and template in blue What do you notice about the structure around the active site (yellow arrow)?

The Y Family of translesion polymerases 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.

10_Table01.jpg DNA repair systems 10

Double strand break repair pathway (homologous recombination) Uses information from homologous sister chromosome and will be discussed next lecture (chapter 11)