The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1,N 6 -ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG. DNA bases are chemically reactive and readily undergo deamination and alkylation on the inevitable exposure to reactive cellular metabolites and environmental toxicants (1-4). Alkylation occurs at many different positions of DNA, producing a variety of lesioned bases (4, 5) that can block replication or interfere with other enzymatic activities templated by DNA. Hypoxanthine is an abundant deaminated base, and it too corrupts the DNA template. Remarkably, human cells appear to produce a single enzyme, alkyladenine DNA glycosylase [AAG (3-methyladenine DNA glycosylase, ANPG, or MPG)], which recognizes and removes hypoxanthine plus a variety of alkylated bases that include 3-methyladenine, 7-methylguanine, and 1,N 6 -ethenoadenine (A; refs. 6 -13). AAG cleaves the N-glycosylic bond joining the damaged base to the DNA backbone, and the resulting abasic nucleotide is excised and replaced with a normal nucleotide by the sequential action of an endonuclease, a polymerase, and DNA ligase (14). The high selectivity for damaged vs. normal bases is essential because normal bases are present in vast excess. AAG can distinguish alternations in both adenine and guanine and can recognize changes present in both the major and minor grooves of DNA. We set out to determine how AAG achieves selectivity for chemically diverse substrates.We previously reported a 2.7-Å crystal structure of AAG complexed to DNA containing a transition-state mimic of the glycosylase reaction, the pyrrolidine abasic nucleotide (pyr; PDB ID code 1bnk; refs. 15 and 16). In the AAG͞pyr-DNA complex, the pyr ring is f lipped into the proposed active site by intercalation of the Tyr-162 side chain into the minor groove of the DNA (15). A bound water molecule in the active site is aligned for a back-side attack of the abasic sugar, but the pyr inhibitor lacks a base, and we could not deduce how AAG recognizes alkylated bases in preference to normal bases. Structures of several other DNA N-glycosylases complexed to their DNA substrates have been reported (17)(18)(19)(20). These enzymes are selective for one type of damaged DNA base and, correspondingly, their active site structures are tailor made for specific interactions with these substrates. For example, uracil DNA glycosylase f lips ur...

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

SUMMARY The NMDA receptor family of glutamate receptor ion channels are formed by obligate heteromeric assemblies of GluN1, GluN2 and GluN3 subunits. GluN1 and GluN3 bind glycine, whereas GluN2 binds glutamate. Crystal structures of the GluN1 and GluN3A ligand-binding domains (LBDs) in their apo states unexpectedly reveal open and closed cleft conformations, respectively, with water molecules filling the binding pockets. Computed conformational free energy landscapes for GluN1, GluN2A, and GluN3A LBDs reveal that the apo state LBDs sample closed cleft conformations, suggesting that their agonists bind via a conformational selection mechanism. By contrast, free energy landscapes for the AMPA receptor GluA2 LBD suggests binding of glutamate via an induced fit mechanism. Principal component analysis reveals a rich spectrum of hinge bending, rocking, twisting, and sweeping motions that are different for the GluN1, GluN2A, GluN3A, and GluA2 LBDs. This variation highlights the structural complexity of signaling by glutamate receptor ion channels.

Most cancer-associated BRCA1 mutations identified to date result in the premature translational termination of the protein, highlighting a crucial role for the C-terminal, BRCT repeat region in mediating BRCA1 tumor suppressor function. However, the molecular and genetic effects of missense mutations that map to the BRCT region remain largely unknown. Using a protease-based assay, we directly assessed the sensitivity of the folding of the BRCT domain to an extensive set of truncation and single amino acid substitutions derived from breast cancer screening programs. The protein can tolerate truncations of up to 8 amino acids, but further deletion results in drastic BRCT folding defects. This molecular phenotype can be correlated with an increased susceptibility to disease. A cross-validated computational assessment of the BRCT mutation data base suggests that as much as half of all BRCT missense mutations contribute to BRCA1 loss of function and disease through protein-destabilizing effects. The coupled use of proteolytic methods and computational predictive methods to detect mutant BRCA1 conformations at the protein level will augment the efficacy of current BRCA1 screening protocols, especially in the absence of clinical data that can be used to discriminate deleterious BRCT missense mutations from benign polymorphisms.Germline mutations within the breast and ovarian cancer susceptibility gene BRCA1 predispose carriers to early-onset breast and breast-ovarian cancers (1). Accumulating evidence points to a role for the BRCA1 protein product in the regulation of multiple nuclear functions including transcription, recombination, DNA repair, and checkpoint control (2-4). Tumor-associated mutations occur throughout the BRCA1 coding sequence, but cluster to sequences encoding the N-terminal RING finger domain and the two carboxy-terminal repeat BRCT 1 domains (5-7).The molecular details of how BRCA1 mutations contribute to the pathogenesis of cancer remain largely unknown. The functional significance of the BRCT region is highlighted by the high degree of sequence conservation within the BRCT regions of among mammalian, Xenopus, and avian BRCA1 homologues (8 -10). Several lines of evidence reveal the BRCT is required for tumor suppressor function. A nonsense mutation, which removes 11 C-terminal residues of the second, BRCT (Tyr 1853 3 stop), is associated with early-onset breast cancer (11). Two cancer-linked BRCT missense mutations (12) that destabilize the BRCT fold (13-15), A1708E and M1775R, ablate the double-strand break repair and transcription function of BRCA1 (16) and inhibit BRCT interactions with histone deacetylases (17), BACH1 (18), and the transcriptional co-repressor CtIP (19,20). Furthermore, mice with homozygous targeted mutations removing the C-terminal half of BRCA1 are viable but develop tumors, suggesting the missing BRCT and/or other domains are expendable for survival, but not for tumor suppression (21).Although all frameshift or nonsense mutations recorded in the Breast cancer Information Cor...