direct reversal (DR)

Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can only occur in one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone.
UV-induced pyrimidine dimers and alkylation adducts can be directly repaired by DNA photolyases and alkyl transferases, respectively.  These repair systems are not followed by incision or resynthesis of DNA.

UV-induced pyrimidine dimers, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, disturb DNA replication and transcription. Some species make use of DNA photolyases to repair these lesions. The FADH- in the photolyase donates an electron to the CPD, which induces the breakage of the cyclobutane bond.
CPD photolyases repair UV-induced CPDs utilizing photon energy from blue or near-UV light. To absorb light, CPD photolyases have two different chromophoric cofactors. One of these, FAD, acts as the photochemical reaction center in the repair process. An electron is transferred from an exogenous photoreductor to FAD, which is changed to the fully reduced, active form FADH-. Although only this chromophore is necessary for the reaction, photolyases have a second chromophore as an auxiliary antenna to harvest light energy, which is transferred to the reaction center. The identity of the second chromophore differs among species. To date, reduced folate (5,10-methenyl-tetrahydrofolate, MTHF), 8-hydroxy-5-deazaflavin (8-HDF), FMN, and riboflavin have been identified as secondary chromophores.
Photolyases usually have a specific binding site for cofactors, but the second chromophore, FMN, of ttPhr shows promiscuous binding with riboflavin or 8-HDF.
Placental mammals lack photoreactivation activity, but they do have nucleotide excision repair (NER) systems for repairing CPDs.

Reversal of O6-Alkylguanine-DNA
O6-alkylguanine is one of the most harmful alkylation adducts and can induce mutation and apoptosis. Almost all species possess mechanisms to repair this adduct. O6-alkylguanine-DNA alkyltransferase (AGT) accepts an alkyl group on a cysteine residue at its active site (PCHR) in a stoichiometric fashion, and this alkylated AGT is inactive. AGT acts as a monomer and transfers the alkyl group from DNA without a cofactor. The structure of human AGT, MGMT, indicates that a helix-turn-helix motif mediates binding to the minor groove of DNA and that O6-methylguanine (O6-meG) is flipped out from the base stack into this active site]. Tyrosine and arginine residues in the active site of the enzyme mediate nucleotide flipping.
The cysteine residue in the active site (PCHR) of AGT is necessary for the methyltransferase activity. Some AGT-like proteins lack cysteine residues in their active sites (PXHR). Alkyltransferase-like (ATL) proteins are a type of AGT homologue and are present in all three domains of life. ATL proteins from E. coli and S. pombe can bind to DNA and show preferential binding to O6-meG-containing DNA, but they are unable to transfer a methyl group from the modified DNA. This binding activity inhibits AGT activity in a competitive manner. E. coli has three AGT homologues, AGT, Ada, and the ATL protein, but S. pombe has only the ATL protein. Therefore, S. pombe is particularly suitable for studies of ATL proteins.
The tyrosine and arginine residues involved in base flipping are also conserved in ATL proteins. The S. pombe ATL protein (Atl1) can bind to the bulky -adduct, O6-4-(3-pyridyl)-4-oxobutylguanine (O6-pobG), with higher affinity than to O6-meG. Additionally, AGT repairs O6-pobG with lower efficiency than O6-meG. In species that have both AGT and ATL protein, for example, E. coli, it is possible that AGT repairs O6-meG while the ATL protein is involved in the repair of bulky O6-adducts such as O6-pobG. 
It is known that the action of ATL proteins is linked with the NER pathway. The ATL protein of E. coli can interact with UvrA and UvrC. Genetic analysis of S. pombe Atl1 showed that atl1 is epistatic to rad13 (the fission yeast orthologue of human ERCC5) and swi10 (the ERCC1 orthologue) but not to rhp14 or rad2 for N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) toxicity. Analyses of the spontaneous mutation rate of rad13 and rad13 atl mutants suggested that ATL-DNA complexes block an alternative repair pathway probably because ATL proteins form a highly stable complex with DNA in the absence of Rad13 or other NER proteins. However, the mechanism by which ATL proteins repair lesions in collaboration with NER proteins is not well understood.  
The protein Ada repairs alkylated lesions in the same manner as AGTs in E. coli. The amino acid sequence and the molecular function of the C-terminal domain of Ada (C-Ada) show similarity to those of AGTs. The N-terminal domain of Ada (N-Ada) can repair a methyl phosphotriester lesion in DNA in vitro. Methylated N-Ada specifically binds to the promoter region of the ada-alkB operon and the alkA and aidB genes and C-Ada can bind to RNA polymerase. Thus, the methylated Ada acts as a transcriptional activator.

Oxidative demethylation by AlkB
AlkB homologues are conserved in many organisms including humans and E. coli. As described above, alkB is one of the genes regulated by Ada. AlkB requires α-ketoglutarate and Fe(II) as cofactors to repair N1-methyladenine or N3-methylcytosine via an oxidative demethylation mechanism. These properties are consistent with the fact that AlkB has sequence motifs in common with 2-oxoglutarate and iron-dependent dioxygenases. AlkB oxidizes the methyl group using nonheme Fe2+, O2, and α-ketoglutarate to restore undamaged bases with subsequent release of succinate, CO2, and formaldehyde. The detailed mechanisms of substrate recognition and catalysis were identified by structural and mutational analyses.
Nine AlkB homologues are known in humans, and, of these, ALKBH1, ALKBH2, ALKBH3, and FTO have been identified as repair enzymes, each of which has a different substrate specificity. E. coli AlkB can repair a lesion in both single-stranded DNA (ssDNA) and dsDNA, whereas ALKBH3 repairs lesions only in ssDNA. ALKBH1 and ALKBH2 can act only on DNA whereas E. coli AlkB and ALKBH3 can act on both DNA and RNA. The crystal structures of AlkB-dsDNA and ALKBH2-dsDNA complexes explain distinct preferences of AlkB homologues for substrates. Cell cycle-dependent subcellular localization experiments suggested that ALKBH2 and ALKBH3 repair mainly newly synthesized DNA and mRNA, respectively, and withhold demethylation of modified rRNA or tRNA.


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ORTHOLOGY CLASS Homo sapiens L. (human) [HSA] Mus musculus L. (mouse) [MMU] Caenorhabditis elegans Maupas (nematode) [CEL] Drosophila melanogaster Meigen (fruit fly) [DME] Saccharomyces cerevisiae Meyen ex E.C. Hansen (budding yeast) [SCE] Schizo-saccharomyces pombe Lindner (fission yeast) [SPO] Escherichia coli Migula (bacterium) K-12 MG1655 [ECO] Arabidopsis thaliana (L.) Heynh. (mouse-ear cress) [ATH]
ko:K10765 (alkylated DNA repair protein alkB homolog 1 [EC:1.14.11.-]) ALKBH1 Alkbh1 AlkB At_AlkB
ko:K02295 (cryptochrome) CRY2
ko:K01669 (deoxyribodipyrimidine photo-lyase [EC:]) TA01342p
Phr1p PhrB CRY3
ko:K10860 (alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 [EC:1.14.11.-]) ALKBH3 Alkbh3
ko:K10859 (alpha-ketoglutarate-dependent dioxygenase alkB homolog 2 [EC:1.14.11.-]) ALKBH2 Alkbh2 At_ALKBH2
ko:K03919 (alkylated DNA repair protein [EC:1.14.11.-]) AlkB
ko:K10778 (AraC family transcriptional regulator, regulatory protein of adaptative response / methylated-DNA-[protein]-cysteine methyltransferase [EC:]) Ada
ko:K00567 (methylated-DNA-[protein]-cysteine S-methyltransferase [EC:]) MGMT Mgmt Y62E10A.5a Dmel_CG1303 Mgt1p Ogt

Last modification date: Oct. 4, 2011