Damage detection and base flipping in direct DNA alkylation repair

Abstract

THE FOREIGN LESION: The mechanistic questions for DNA base damage detection by repair proteins are discussed in this Minireview. Repair proteins could either probe and locate a weakened base pair that results from base damage, or passively capture an extrahelical base lesion in the first step of damage searching on double-stranded DNA. How some repair proteins, such as AGT (see figure), locate base lesions in DNA is still not fully understood.To remove a few damaged bases efficiently from the context of the entire genome, the DNA base repair proteins rely on remarkably specific detection mechanisms to locate base lesions. This efficient molecular recognition event inside cells has been extensively studied with various structural and biochemical tools. These studies suggest that DNA base damage can be located by repair proteins by using two mechanisms: a repair protein can probe and detect a weakened base pair that results from mutagenic or cytotoxic base damage; alternatively, a protein can passively capture and stabilize an extrahelical base lesion. Our chemical and structural studies on the direct DNA repair proteins hAGT, C-Ada and ABH2 suggest that these proteins search for weakened base pairs in their first step of damage searching. We have also discovered a very unique base-flipping mechanism used by the DNA repair protein AlkB. This protein distorts DNA and favors single stranded DNA (ssDNA) substrates over double-stranded (dsDNA) ones. Potentially, it locates base lesions in dsDNA by imposing a constraint that targets less rigid regions of the duplex DNA. The exact mechanism of how AlkB and related proteins search for damage in ssDNA and dsDNA still awaits further studies.

Figure 1

Cartoon presentation of selected base-specific repair proteins bound to dsDNA. Proteins are colored green, flipped-out bases are magenta, finger residues are blue, and DNA is bright orange. Escherichia coli AlkB (PDB ID 3BIE) is of particular uniqueness in the way of flipping out a 1-meA damaged base into the active pocket lacking a finger residue. Human AAG (PDB ID 1EWN), AGT (PDB ID 1T38), OGG (PDB ID 1EBM), UDG (PDB ID 4SKN), and ABH2 (PDB ID 3BUC) proteins flip εA, O⁶-methylG, 8-oxo-guanine, uracil, and 1-methyladenine out of the DNA helix into specific recognition pockets by intercalating a finger residue into the double helix, respectively.

Figure 2

Proposed damage searching and base flipping mechanisms for DNA repair proteins. a In this model, the protein actively flips every base out and checks it in its active site pocket until the lesion is located. b Proposed damage searching model by detecting weakened or non Watson-Crick base pairs. The repair protein would distort the double helical structure of DNA and selectively flip out an unstable base. c DNA repair protein passively captures a transiently extrahelical lesion.

Figure 3

DNA bases pairing of Hoogsteen type T·1-meA, Wobble type O⁶meG·C, and the normal Watson-Crick O⁶meG·T.

Figure 4

A disulphide cross-linking strategy of C-Ada protein with different dsDNAs to investigate damage detection and base flipping. a C-Ada uses reactive Cys139 residue to selectively transfer the alkyl modification from O⁶-alkylatedguanine to itself, which represents a suicidal direct repair. b A thiol-tethered cytosine was introduced in a cross-linking reaction between protein and DNA. c Various DNA probes, including ssDNA and dsDNA were used in this study. d The cross-linking results were shown by non-reduced SDS-PAGE. e Effect of external thiol (DTT) on the cross-linking reactions.

Figure 5

A structure of the human AGT/DNA complex. a A monomer AGT protein binds at the junction of the two DNA fragments, and recognizes a terminal overhanging thymine. This base is inserted into the active site pocket only partially rather than forming a base pair with the adenine overhang from the adjacent DNA. b Cartoon presentation of the AGT protein binding the junction of two duplex DNA. The finger residue, Arg128 that intercalates inside the duplex and fills the gap left by base flipping, is shown.

Figure 6

Cartoons for dsDNA conformations in the presence and absence of bound AlkB or ABH2. a 1-meA may adopt an intrahelical configuration in double helix by forming a Hoogsteen type base paring with thymine from the complementary strand. b Escherichia coli AlkB squeezes dsDNA and induces severe distortion of the DNA duplex to facilitate base flipping. c ABH2 is a standard dsDNA damage repair protein that uses a finger residue (Phe102) to stabilizes the duplex structure after base flipping. d A close view of the structure of how AlkB binds dsDNA. e A close view of how ABH2 binds dsDNA