5-methylcytosine recognition by Arabidopsis thaliana DNA glycosylases DEMETER and DML3.

Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA binding to identify residues important for recognition of 5mC within the context of DNA and inside the enzyme active site. Our results indicate that the 5mC binding pocket is composed of residues from discrete domains and is responsible for discrimination against 5mC derivatives, and suggest that DME, ROS1, and DML3 utilize subtly different mechanisms to probe the DNA duplex for cytosine modifications.

Recent advances in the structural mechanisms of DNA glycosylases.

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities.

Interactions of actinomycin D with human telomeric G-quadruplex DNA.

The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticancer drug discovery. The human telomeric G-quadruplex consists of a single strand repeat of d[AGGG(TTAGGG)(3)] that can fold into higher-order DNA structures. Small molecules that selectively target and stabilize the G-quadruplex structure(s) may serve as potential therapeutic agents and have garnered significant interest in recent years. In the work presented here, the anticancer agent, actinomycin D, is demonstrated to bind to and induce changes in both structure and stability in both the Na(+) and K(+) forms of the G-quadruplex DNA. The binding of actinomycin D to the G-quadruplex DNAs is characterized by intrinsic association constants of approximately 2 x 10(5) M(-1) (strand) and 2:1 molecularity, and are shown to be enthalpically driven with binding enthalpies of approximately -7 kcal/mol. The free Na(+) or K(+) forms of the quadruplex structures differ in melting temperatures by approximately 8 degrees C (60 and 68 degrees C, respectively), whereas both forms, when complexed with actinomycin D are stabilized with melting temperatures of approximately 79 degrees C. The induced CD signals observed for the actinomycin D-G-quadruplex complexes may indicate that the phenoxazone ring of actinomycin D is stacked on the G-tetrad rather than intercalated between adjacent G-tetrads. Complex formation with actinomycin D results in changes to both the Na(+) or K(+) structural isoforms to ligand-bound complexes having similar structural properties and stabilities.