Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A1-4 is an endogenous mutagen in many different human cancers5-7, where it becomes hijacked to fuel tumor evolvability. APOBEC3A's single-stranded DNA C-to-U editing activity1,8 results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations5-7. Transgenic expression in mice demonstrates its tumorigenic potential9. APOBEC3A inhibitors may therefore comprise a novel class of anti-cancer agents that work by blocking mutagenesis, preventing tumor evolvability, and lessening detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC recognition motif that is part of a three-nucleotide loop. The nuclease-resistant phosphorothioated derivatives of these inhibitors maintain nanomolar in vitro potency against APOBEC3A, localize to the cell nucleus, and block APOBEC3A activity in human cells. These results combine to suggest roles for these inhibitors to study A3A activity in living cells, potentially as conjuvants, leading toward next-generation, combinatorial anti-mutator and anti-cancer therapies.

α-2'-Deoxyguanosine can switch DNA G-quadruplex topologies from antiparallel to parallel.

Here we demonstrate G-quadruplex formation by oligodeoxynucleotides containing α-2'-deoxyguanosine (α-dG) as a sole source of guanosines in G4T4, G4T4G4 and T(G3Tn)3G3T sequences with various numbers of natural ß-T in the loops (n = 1-4). Based on circular dichroism spectra we observed that all α-dG-containing DNAs formed G-quadruplexes with uniform arrangement of α-dG-tetrads, which implies formation of G-quadruplexes of parallel topology. In several cases, native DNA structures that usually adopt an antiparallel topology were converted to more thermally stable G-quadruplexes of parallel topology. Using 2D ROESY NMR spectra a new 'sequential walk' was established for α-dGs in a tetramolecular, parallel complex formed by the α-G4ß-T4 sequence. Analysis of ROEs in α-dGs indicates that guanines in [α-G4ß-T4]4 adopt anti-glycosidic conformations. These results demonstrate that α-dG can be used for an antiparallel-to-parallel switch of G-quadruplex DNAs producing complexes with higher thermal stability and uniform stacking of α-dG-tetrads.

Identification of a periplasmic AlgK-AlgX-MucD multiprotein complex in Pseudomonas aeruginosa involved in biosynthesis and regulation of alginate.

The opportunistic human pathogen Pseudomonas aeruginosa produces an extracellular polysaccharide called alginate. This is especially relevant in pulmonary infection of cystic fibrosis patients where it protects the bacteria from the hosts' immune system and the diffusion of antibiotics. Here a connection between the stability of a proposed alginate polymerisation/secretion complex and the regulation of the operon encoding these proteins was assessed. Experimental evidence was provided for a periplasmic multiprotein complex composed of AlgX, AlgK, and the regulatory protein MucD. Disruption of the alginate machinery in a mucoid strain, either by removal, or over production of various essential proteins resulted in an at least 2-fold increase in transcription of a lacZ reporter under the control of the algD promoter. Instability of the complex was indicated by an increase in secretion of alginate degradation products. This increase in transcription was found to be dependent on the negative regulatory protein MucD. Surprisingly, over production of MucD leads to a 3.3-fold increase in transcription from the alginate promoter and a 1.7-fold increase in the levels of alginate produced, suggesting an additional positive regulatory role for MucD in mucoid strains. Overall, this study provided experimental evidence for the proposed periplasmic multiprotein complex and established a link of a constituent of this complex, MucD, to transcriptional regulation of alginate biosynthesis genes.