Structural snapshots of base excision by the cancer-associated variant MutY N146S reveal a retaining mechanism.

DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated ß-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision.

Structural Basis for Finding OG Lesions and Avoiding Undamaged G by the DNA Glycosylase MutY.

The adenine glycosylase MutY selectively initiates repair of OG:A lesions and, by comparison, avoids G:A mispairs. The ability to distinguish these closely related substrates relies on the C-terminal domain of MutY, which structurally resembles MutT. To understand the mechanism for substrate specificity, we crystallized MutY in complex with DNA containing G across from the high-affinity azaribose transition state analogue. Our structure shows that G is accommodated by the OG site and highlights the role of a serine residue in OG versus G discrimination. The functional significance of Ser308 and its neighboring residues was evaluated by mutational analysis, revealing the critical importance of a ß loop in the C-terminal domain for mutation suppression in cells, and biochemical performance in vitro. This loop comprising residues Phe307, Ser308, and His309 (Geobacillus stearothermophilus sequence positions) is conserved in MutY but absent in MutT and other DNA repair enzymes and may therefore serve as a MutY-specific target exploitable by chemical biological probes.

Co-evolution of SNF spliceosomal proteins with their RNA targets in trans-splicing nematodes.

Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2B″/SNF family (hereafter referred to as the SNF family) of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). The highly constrained nature of this system has inhibited an analysis of co-evolutionary trends between the proteins and their RNA binding targets. Here we report accelerated sequence evolution in the SNF protein family in Phylum Nematoda, which has allowed an analysis of protein:RNA co-evolution. In a comparison of SNF genes from ecdysozoan species, we found a correlation between trans-splicing species (nematodes) and increased phylogenetic branch lengths of the SNF protein family, with respect to their sister clade Arthropoda. In particular, we found that nematodes (~70-80 % of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. Interestingly, this increased evolutionary rate correlates with the reliance on trans-splicing by nematodes, which would alter the role of the SNF family of spliceosomal proteins. We mapped amino acid substitutions to functionally important regions of the SNF protein, specifically to sites that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF's RNA targets: the U1 and U2 snRNAs. Both are more divergent in nematodes than arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.