CH 11: DNA Repair

DNA Damage
Lesion = chemical damage in DNA Chemicals that cause lesion can kill the bacterium
Increase frequency of mutations

Kill curves (Survival curve)

Expose cells to a chemical plate cells are various exposure times.

Fig. 11.1 Survival Curve

Types of Mutations
Deaminations Oxygen damage Alkylation Pyrimidine dimers

Deamination of Bases
Amino groups in adenine, cytosine and guanine are removed. Adenine = hypoxanthine Guanine = xanthine Cytosine = uracil Mutagenic because it cases mispairing

Fig. 11.2: Modified Bases

Normal Base Pairing

Consequences of Deamination
A = hypoxanthine =looks like a G
Now base pair to a C

G = xanthine
Still base pair with a C

C = uracil = looks like a T

Base pair with an adenine

Deaminating Agents
Hydroxylamine (cant enter cells-use in vitro)
GC to AT only Modifies C only

Bisulfite
Only C

Nitrous acid
Removes amino groups from all bases

Repair of Deaminated Bases

DNA glycosylases
Break glycosyl bond between damaged bases and sugar nucleotide Specific glycosylases for each base

Fig. 11.3 Repair by glycosylases

Fig. 3.11: Removal of deaminated cytosine

Fig. 11.2b

Very Short Patch Repair

Deamination of methyl-cytosine = thymine
How do we know this is wrong This is old DNA cant use unmethylated Vs. methylated DNA

Location important
Most methyl-C is 2nd C of sequence 5 CCWGG 3

VSP
Endonuclease = Vsr endonuclease
Binds to TG mismatch Makes a break next to the T DNA polymerase I removes the T and resynthesizes the DNA

Very specific Gene for Vsr endonuclease downstream of dcm methylase

Damage due to Oxygen

Free radicals, superoxide radicals All aerobic organisms have this problem 7,8-dihydro-8-oxoguanine (8-oxoG or GO)

MutM, MutY, MutT

Preventing mutations from GO MutM = N-glycosylase that removes GO MutY = removes adenines that have been incorporated opposite GO MutT = prevents GO from entering DNA

Fig. 11.4 Mechanism for Avoiding Mutagenesis from GO

Genetics of GO Mutagenesis
If MutT mutated, more GO in DNA If MutM doesnt remove GO, spontaneous mutation rate higher. If MutY doesnt remove the As opposite GO, even higher spontaneous mutation rate.

Alkylation
Base and phosphate can be alkylated
Ethyl methanesulfonate (EMS) Nitrosogianidine

N7-guanine and N3 of adenine are targets

Severely alter pairing Distort helix

Fig. 11.5: Alkylation of G

Removing Alkylated Bases

N-glycosylases = alkA in E.coli Methyltransferases
Remove alkyl group Ogt =actively growing cells Ada = stationary cells

Adaptive Response
Induction of genes necessary for repair of alkylation After exposure to alkylating agents cells resistant to more treatments
Ada genes or N-glycosylases

Fig. 11.6a Regulation of Adaptive Response

aidB ada alkB
Ada

alkA

alkB

Fig. 11.6b Regulation of Adaptive Response

aidB ada alkB
Ada
P-Me

alkA

alkB

Ada

G-Me

Suicide inactivation
Me

Me

Pyrimidine Dimers
UV irradiation = natural damage to DNA Conjugated-ring structure
Abnormal linkages between bases

Fig. 11.7 Two Common Types of Pyrimidine Dimers

Fig. 11.8: Photoacti vation