Biochemical and structural studies reveal the substrate specificity and catalytic mechanism of MYG1 as a two-metal ion-dependent 3′→5′ exonuclease

Abstract

Nucleases are a class of enzymes that specifically cleave nucleic acids in all living organisms. They play crucial roles in essential biological processes, including the regulation of gene expression, DNA damage repair, and RNA processing and degradation. MYG1 (melanocyte proliferating gene 1) is a highly conserved eukaryotic protein that exhibits 3′→5′ exonuclease activity. This study systematically characterizes the enzymatic properties of MYG1 and determines its structures in complexes with metal ions and various mono- and poly-(deoxy)nucleotides. The functional roles of key residues involved in metal ion binding and substrate binding in the catalytic reaction are examined through site-directed mutagenesis, enzymatic activity assay, and structure determination. Our biochemical and structural data together demonstrate that MYG1 is a Mn2+- or Mg2+-dependent 3′→5′ exonuclease capable of cleaving a variety of nucleic acids with different structures. It exhibits the highest activity for single-stranded RNA and a nucleotide preference for U in single-stranded RNA and dT in single-stranded DNA. Mechanistically, MYG1 functions as a dimer, with the active site formed by the catalytic domain of monomer 1 and the substrate-binding domain of monomer 2, and cleaves nucleic acids through a two-metal ion-mediated catalytic mechanism. These findings establish a molecular basis for further investigations into the biological functions and molecular mechanisms of MYG1 within cells and its potential roles in human diseases.

1. Introduction

Nucleic acids are fundamental macromolecules of life, including DNA and RNA, which together are responsible for the storage and transmission of genetic information. Nucleases are enzymes that specifically recognize and cleave phosphodiester bonds in nucleic acids. Thus, they play a critical role in the metabolism of nucleic acids and are involved in various biological processes, including the regulation of gene expression, DNA damage repair, and RNA processing and degradation [1]. Nucleases can be classified into endonucleases and exonucleases based on their mode of action and deoxyribonucleases and ribonucleases according to their substrate preference [2–4]. They also include specialized categories such as topoisomerases, recombinases, ribozymes, and RNA splicing enzymes. From the perspective of the catalytic mechanism, nucleases can be divided into three major classes: two-metal ion dependent, one-metal ion dependent, and metal ion independent. Nevertheless, within the same mechanistic class, members can exhibit significant differences in three-dimensional structures, metal ion coordination patterns, and biological functions [5]. Nucleases often possess modular domain architectures, achieving efficient recognition and cleavage of specific nucleic acid sequences through the coordinated action of functional domains such as catalytic domains, nucleic acid-binding domains, and recognition domains [5].

DHH family nucleases are a group of evolutionarily highly conserved and functionally diverse nucleases, which are found widely across bacteria, archaea, and eukaryotes [6,7]. Members of this family share a highly conserved DHH (Asp-His-His) motif within the catalytic domain, which, together with an adjacent substrate-binding domain, forms the active site. The catalytic activity strictly depends on divalent metal ions such as Mn2+ or Mg2+ and typically employs a two-metal ion-dependent catalytic mechanism. Despite their conserved catalytic core, DHH family nucleases exhibit highly diverse substrate specificity and generally lack strict sequence-specific recognition. As a result, they fulfill a wide range of functional roles and are involved in a variety of biological processes, including DNA repair and recombination, RNA maturation and degradation, stress tolerance, cell signaling, and immune regulation [7–10]. Additionally, dysregulation or dysfunction of DHH family nucleases can result in abnormal RNA processing and genomic instability, leading to their implication in various diseases, including cancers and genetic disorders [11–13].