DNA Repair and Mutations. Chemical reactions and some physical processes constantly damage genomic DNA At the molecular level, damage usually involves changes in the structure of one of the strands Vast majority are corrected by repair systems using the other strand as a template
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Chemical reactions and some physical processes constantly damage genomic DNA
At the molecular level, damage usually involves changes in the structure of one of the strands
Vast majority are corrected by repair systems using the other strand as a template
Some base changes escape repair and the incorrect base serves as a template in replication
The daughter DNA carries a changed sequence in both strands; the DNA has been mutated
Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens
Mismatches arise from occasional incorporation of incorrect nucleotides
Abnormal bases arise from spontaneous deamination reactions or via chemical alkylation (alk genes)
Pyrimidine dimers form when DNA is exposed to UV light
Backbone lesions occur from exposure to ionizing radiation
Figure 30-51Types and sites of chemical damage to which DNA is normally susceptible in vivo. Red, oxidation; blue, hydrolysis; green, methylation.
Endogenous and exogenous alkylating agents (tobacco smoke, some anticancer drugs). Alkylation destabilizes the glycoside bond and can ultimately lead to backbone breaks.
O6-alkylguanine has a different pattern of H-bond donor and acceptor atoms than the parent guanine base. As a result, it base pairs with T instead of C, giving rise to G A transition after the second round of replication:
O6-alkylguanine DNA alkyltransferase (AGT)
Directly repaires alkylation damage (O6-alkylguanines) by transferring the O6-alkyl group from damaged guanine in DNA to a Cys residue in the AGT active site in a stoichiometric reaction. The protein is inactivated via alkylation and undergoes proteolytic degradation.
AGT protein is highly conserved:
helix-turn-helix DNA binding motif
the alkylated base is “flipped” out of the helix to enter the hydrophobic alkyl-binding pocket of the protein
high metabolic cost for the cell is outweighed by the need to maintain genetic integrity
Formation of pyrimidine dimers induced by UV light. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA
Photolyase: repairs cyclobutane pyrimidine dimers. Uses the energy of light to catalyze the reversal of the cyclobutane bonds, producing intact DNA. Not very important in mammals.
Nucleotide-excision repair in E. coli and humans
Base-excision repair pathway
1 DNA glycosylase recognizes damaged base, cleaves N-glycoside bond
2 AP endonuclease cleaves backbone near the AP site.
3 DNA pol I initiates repair synthesis from the free 3′ OH at the nick, removing and replacing the damaged strand.
4 Nick sealed by DNA ligase.
Intermediates on the base flipping pathways of Human 8-oxoguanine glycosylase hOGG1) The exo-site complex of hOGG1 with an extrahelical guanine (by chemical trapping)(left). The fully extrahelical complex with 8-oxoG is shown on the right.
Note the DNA distortion in the NON substrate.
Extrahelical Damaged Base Recognition by DNA Glycosylase Enzymes
Chemistry - A European Journal
Volume 14, Issue 3, pages 786–793, January 18, 2008
X-Ray structure of human uracil–DNA glycosylase (UDG) in complex with a 10-bp DNA containing a U·G base pair. Mismatch flipped out. DNA helix stabilized by aa plug.
Catalysis is by GAB, but activity is not abolished by loss of the acid residue in the active site. Conformational strain helps drive the reaction!!
aa plug prevents base from flipping back into helix.
Base excision repair of alkylated DNA by AlkD in Bacillus cereus.
Lack of +
Resembles apurinc DNA
a, AlkD catalyses the hydrolysis of the N-glycosidic bond to liberate an abasic site and free nucleobase (100X rate increase over spontaneous depurination). The enzyme is specific for positively charged N3-methyladenine (a) and N7-methylguanine (b). c, d, Structures used to trap AlkD in complex with alkylated and abasic DNA. e, Crystal structure of AlkD bound to 3d3mA-DNA. Each of the six HEAT repeats is coloured red-to-violet. The DNA is coloured silver with the 3d3mA nucleotide colored magenta.
EH Rubinson et al.Nature000, 1-6 (2010) doi:10.1038/nature09428
Figure S3. DNA binding by the HEAT repeats of AlkD. Electrostatic surface potential (blue, positive; red, negative) of AlkD showing a high degree of positive charge within the concave DNA binding cleft. Structure based sequence alignment of HEAT repeats. Residues that contact the DNA in both substrate (3d3mA•T, G•T) and product (THF•T, THF•C) complexes are highlighted yellow, residues contacting the DNA in only substrate or only product are highlighted blue and magenta, respectively.
Crystal structures of AlkD in complex with 3d3mA-DNA (a) and THF-DNA (b).
EH Rubinson et al.Nature000, 1-6 (2010) doi:10.1038/nature09428
The modified 3d3mA and tetrahydrofuran (THF) nucleotides are colored blue, and the opposing thymine is magenta.
DNA positioned along +charged concave surface of alkD
Contacts 10 bp
Contacts cluster around mismatch
Fewer contacts on lesion strand
Both 3m3A and THF reside on the face of the DNA duplex NOT in contact with the protein whereas the compelement is nestled in the enzyme cleft.
This THF trapped complex shows the abasic site rotated 90o around the PD backbone where it is totally solvent exposed!!
Opposing T has slipped out of helix into DNA minor groove!!
NO aa PLUG
DNA duplex has collapsed to retain stacking interactions.
Recognition of DNA damage by AlkD.
a, 3d3mA-DNA (substrate) complex.
b, THF-DNA (product) complex.
Dashed arrows denote displacement of THF and opposing thymine from their positions in B-DNA. Hydrogen bonds are shown as dashed lines. Views are down the DNA helix axis.
Remodelling of a G•T wobble base pair by AlkD.
a, AlkD–G•T-DNA complex viewed down the helical axis. b, The structure of a G•T wobble base pair in DNA alone is superimposed onto the AlkD–G•T complex. Steric clashes between the protein and DNA are highlighted by yellow stars, and disrupted hydrogen bonds (dashed lines) are shown by a red X. c, Relative single-turnover rates (kst) of 7mG excision from a 25mer oligonucleotide duplex by wild-type AlkD and the indicated AlkD mutants.
AlkD seems to detect DNA duplex destabilization rather than specifically recognizing modified bases. Protein restructures the wobble and disrupts base stacking.
Solvent exposure increases the lifetime that spontaneous depurination is likely to occur!!!The phosphate groups on the DNA may participate in the rate enhancement by positioning water molecules for solvent attack on the glycoside bond.
AlkD captures the DNA in an orientation that holds the orphaned base next to the protein and exposes the lesion to a hydrolytic environment. The distorted DNA conformation is stabilized not by a side chain plug, but by stacking of flanking base pairs as a result of both lesion and orphaned base flipping. Phosphate (gold “P”) assisted hydrolysis could occur either by positioning of the water molecules adjacent to the C1' carbon in a dissociative hydrolysis reaction, or through stabilization of an oxocarbenium ion intermediate
Figure S12. Proposed mechanism for how AlkD facilitates hydrolysis of N3 and N7-alkylpurines by distorting the DNA backbone.
Methylation and mismatch repair
Really only understood well in E.coli.
The methylation occurs at the N6 of adenines in (5′)GATC sequences.
Dam=DNA adenine methylation
This ensures that DNA damages are repaired before the cell divides