Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.Cellular DNA is constantly subjected to modifications by environmental and endogenous chemicals, which can result in covalent changes 1,2 . Methylating (or alkylating) agents are a common group of DNA modifiers that introduce damage primarily to the heterocyclic bases of DNA, with mutagenic and/or cytotoxic consequences. Alkylating agents are also widely used in cancer therapy and exert anticancer effects by creating cytotoxic DNA lesions in tumour cells. Many of these alkylation DNA damages are detected and repaired by proteins that are conserved across kingdoms.The E. coli AlkB protein is a direct dealkylation DNA repair protein 3-5 . It uses a mononuclear iron(II) site and cofactors 2-ketoglutarate (2KG) and dioxygen to perform an unprecedented oxidative demethylation of DNA base lesions 1-meA, 3-meC, 1-meG and 3-meT ( Supplementary Fig. 2) 6-11 . AlkB also removes etheno DNA lesions by using a similar oxidation mechanism 12,13 . There are nine potential human homologues of AlkB. Two of Correspondence and requests for materials should be addressed to C.H. (chuanhe@uchicago.edu). * These authors contributed equally to this work. Reprints and permissions information is available at www.nature.com/reprints. Author Contributions NIH Public Access Cross-linking to stabilize protein-DNA complexesWe report here the first crystal structures of AlkB-dsDNA and ABH2-dsDNA complexes. The AlkB family proteins bind DNA weakly 21 and form labile complexes with damagecontaining DNA 22 , which makes crystallization of their protein-DNA complexes challenging.To overcome this difficulty we used chemical cross-linking methods 23,24 ; initially using an active site disulphide cross-linking strategy that we developed previously (Fig. 1a) 25,26 . Baserepair proteins flip damaged bases and insert them into the active site for processing. Therefore, we reasoned, a cysteine residue engineered into the active site of AlkB may form a disulphide cross-link, at equilibrium, with a disulphide-modified cytosine (C* in a C*:A base pair) flipped into the active site of the repair protein ( Fig. 1a) 27 ....

Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections. The protein MgrA is both an important virulence determinant during infection and a regulator of antibiotic resistance in S. aureus. The crystal structure of the MgrA homodimer, solved at 2.86 A, indicates the presence of a unique cysteine residue located at the interface of the protein dimer. We discovered that this cysteine residue can be oxidized by various reactive oxygen species, such as hydrogen peroxide and organic hydroperoxide. Cysteine oxidation leads to dissociation of MgrA from DNA and initiation of signaling pathways that turn on antibiotic resistance in S. aureus. The oxidation-sensing mechanism is typically used by bacteria to counter challenges of reactive oxygen and nitrogen species. Our study reveals that in S. aureus, MgrA adopts a similar mechanism but uses it to globally regulate different defensive pathways.

Introduction Overview of Direct Repair of DNA Alkylation DamageCellular DNA is constantly subjected to modifications by intracellular and extracellular chemicals, which can result in covalent changes. 1,2 Alkylating agents are one group of such chemicals that can lead to DNA damage. 3 These agents are prevalent in the environment and are used as anticancer compounds in the clinical setting. 4-10 Alkylating agents also exist endogenously inside cells; for instance, S-adenosylmethionine, a methyl donor for many cellular reactions, has been shown to produce methylation damage. 11,12 The attack on DNA by these alkylating agents can lead to various types of lesions on the heterocyclic bases or backbone. 3,13-15 Most of these resulting adducts are mutagenic or toxic, and cells have evolved various proteins to detect and repair them. 9,16,17 Interestingly, many of these alkylation lesions are repaired through the direct removal of the adduct. Other than the photolyase that catalyzes direct reversal of the thymine dimer created by UV light, 18,19 all known direct DNA repair proteins are engaged in alkylation DNA damage repair. These are the N-terminal domain of the Escherichia coli (E. coli) Ada protein, the O 6 -alkylguanine-DNA alkyltransferase family, and the AlkB family. 9 1.1.1. Alkylation of DNA-Alkylating reagents can be divided into S N 1 and S N 2 types based on the mechanism of the alkylation attack. The alkylation susceptibility of each site on the bases or backbone varies depending on the reagent used (Figure 1); the resulting lesions also have different mutagenic and cytotoxic effects. The N 7 -position of guanine is the most vulnerable site on DNA; unsurprisingly, it also serves as the best ligand on the DNA for metal ions such as platinum(II). 20 Treating double-stranded DNA (dsDNA) with methylating agents such as methylmethane sulfonate (MMS, an S N 2 type methylating agent) or N-methyl-N′nitrosourea (MNU, an S N 1 type methylating agent) typically results in 70-80% of the methylation occurring on the N 7 -position of guanine. Despite being the most abundant product of alkylation damage, N 7 -methylguanine is relatively innocuous and is removed mostly through spontaneous depurination. 21 The resulting abasic site is toxic and repaired enzymatically. 22 The N 3 -methyladenine is the second most abundant alkylation lesion formed in dsDNA. This lesion can block DNA replication and is removed by AlkA in E. coli and 3methyladenine-DNA-glycosylases. [23][24][25] The S N 1 type methylating agents such as MNU and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) are highly mutagenic because they attack the oxygen atoms on DNA bases to give a significant amount of O 6 -methylguanine (O 6 -meG) and a small amount of O 4 -methylthymine (Figure 1). 13,14 O 6 -meG mispairs with thymine during DNA replication, which gives rise to a transition mutation of G:C to A:T. 26-29 Thus, this lesion must be rapidly located and removed in order to maintain the integrity of the genome. The O 6 -alkylguanine-DNA alkyltransferase family o...

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RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab:ligase complex at 3.1Å using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone’s role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix and retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of the smaller size of the loop and high molecular weight, large surface area, and phasing power provided by Fabs.