Recognition of single-stranded DNA by nuclease P1: High resolution crystal structures of complexes with substrate analogs

Abstract

The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 A structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspergillus oryzae are discussed.

A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA

“…We therefore developed a complementary method to identify nucleotides conjugated to base-labile or nucleophile-labile small molecules. In this second method, the macromolecular fraction of whole cellular RNA was treated with nuclease P1, an endonuclease that cleaves RNA to generate mononucleotides with a 3Ј-hydroxyl group and a 5Ј-phosphate (29). As in the first method, one half of the resulting sample was treated with base (pH 10.5) or nucleophile (500 mM n-butylamine in acetonitrile) and the other half was treated with control conditions (pH 4.5, or acetonitrile, respectively).…”

“…To test this hypothesis we digested total RNA from both E. coli and S. venezuelae with nuclease P1 in the presence of 18 O-enriched water. Because nuclease P1 catalyzes the attack of a water molecule on RNA to generate 5Ј-phosphonucleotides (29), in the presence of 18 O water all nuclease P1 digestion products other than the nucleotides at the 5Ј termini will have a mass shift of ϩ2 Da compared with products of digestion in 16 O water. Indeed, the expected ϩ2 Da shift was observed for 3Ј-Phe-AMP (observed [MϪH] Ϫ m/z ϭ 495.1290; expected [MϪH] Ϫ m/z ϭ 495.1285).…”

Photocrosslinking of human telomeric G-quadruplex loops by anti cyclobutane thymine dimer formation

“…NP1 is an endonuclease that degrades photoproduct-containing DNA to smaller photoproduct-containing fragments that are characteristic of the type of photodamage (20,22). NP1 functions by binding to the base portion of a nucleotide after which it hydrolyzes the phosphodiester bond on the 3Ј-side to yield a deoxynucleotide with a 3Ј-hydroxyl group (23,24). Because NP1 does not bind to photodimerized bases, it cannot cut on the 3Ј-side of either nucleotide involved in photodimer formation.…”

Dissection of the Functional Domains of theLeishmania Surface Membrane 3′-Nucleotidase/Nuclease, a Unique Member of the Class I Nuclease Family

“…Although the three-dimensional structure of the P1 nuclease has been determined (8) and a putative mechanism of catalysis has been proposed (9), no structure/function studies have been performed with any other member of the class I nuclease family. In this report, we used a homologous leishmanial expression system to dissect and analyze the functional domains of the parasite 3Ј-nucleotidase/nuclease.…”