Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer

Abstract

Sterile Alpha Motif and HD Domain Protein 1 (SAMHD1) is a unique enzyme that has important roles in nucleic acid metabolism, viral restriction, and the pathogenesis of autoimmune diseases and cancer. Although much attention has been focused on its dNTP triphosphohydrolase activity in viral restriction and disease, SAMHD1 also binds to single-stranded RNA and DNA. Here we utilize a UV crosslinking method using 5-bromodeoxyuridine-substituted oligonucleotides coupled with high-resolution mass spectrometry (HRMS) to identify the binding site for single-stranded nucleic acids (ssNA) on SAMHD1. Mapping cross-linked amino acids on the surface of existing crystal structures demonstrated that the ssNA binding site lies largely along the dimer-dimer interface, sterically blocking the formation of the homotetramer required for dNTPase activity. Surprisingly, the disordered C-terminus of SAMHD1 (residues 583–626) was also implicated in ssNA binding. An interaction between this region and ssNA was confirmed in binding studies using the purified SAMHD1 583–626 peptide. Despite a recent report that SAMHD1 possesses polyribonucleotide phosphorylase activity, we did not detect any such activity in the presence of inorganic phosphate, indicating that nucleic acid binding is unrelated to this proposed activity. These data suggest an antagonistic regulatory mechanism where the mutually exclusive oligomeric state requirements for ssNA binding and dNTP hydrolase activity modulate these two functions of SAMHD1 within the cell.

SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

“…Our finding of a rescue of the HR and DNA end resection impairment of SAMHD1 depletion with an active-site mutant of SAMHD1, which abolishes its dNTPase activity and reported nuclease activity, and interaction of SAMHD1 with CtIP/MRE11, which are known nucleases, suggest that SAMHD1’s role in promoting DNA end resection is likely independent of any intrinsic catalytic activity and thus through a scaffold function. In this respect, SAMHD1 localizes to naDNA at CPT-induced DSBs and binds to ssDNA/RNA (Beloglazova et al, 2013; Goncalves et al, 2012; Seamon et al, 2016; Seamon et al, 2015; Tungler et al, 2013) and could facilitate CtIP recruitment to or stabilization at DSBs through this interaction. CtIP recruitment to DNA damage sites is also dependent on its interaction with the MRN complex (You et al, 2009; Yuan and Chen, 2009), BRCA1 (Yu et al, 2006), and its own tetramerization (Wang et al, 2012), which could be regulated by SAMHD1.…”

“…SAMHD1 binds to ssDNA at its dimer-dimer interface, which sterically blocks its tetramerization into its dNTPase active form (Brandariz-Nunez et al, 2013; Hansen et al, 2014; Ji et al, 2014; Seamon et al, 2016; Yan et al, 2013; Zhu et al, 2013). Thus, SAMHD1 may function as a monomer or dimer in promoting DNA end resection and tetramer in promoting dNTP metabolism, which is controlled by its binding to ssDNA in response to DNA DSB induction.…”

“…SAMHD1 contains a SAM domain, a protein interaction module (Schultz et al, 1997) and a histidine-aspartic acid (HD) domain, found in a superfamily of proteins with metal dependent phosphohydrolase activity (Aravind and Koonin, 1998). In addition to its well-established dNTPase activity, SAMHD1 binds to ssDNA/RNA (Beloglazova et al, 2013; Goncalves et al, 2012; Seamon et al, 2016; Seamon et al, 2015; Tungler et al, 2013) at its dimer-dimer interface, which sterically blocks tetramerization (Seamon et al, 2016) required for its dNTPase activity (Brandariz-Nunez et al, 2013; Hansen et al, 2014; Ji et al, 2014; Yan et al, 2013; Zhu et al, 2013), and SAMHD1 has been reported to possess DNase/RNase activity (Beloglazova et al, 2013; Ryoo et al, 2014); however, a number of studies indicate that SAMHD1 lacks active-site associated nuclease activity (Antonucci et al, 2016; Goldstone et al, 2011; Goncalves et al, 2012; Seamon et al, 2016; Seamon et al, 2015; Welbourn and Strebel, 2016), which has been attributed to persistent co-purifying contaminants (Antonucci et al, 2016; Seamon et al, 2015). …”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…Efficient interaction with ssNAs involve residues from both the HD domain and C-terminus, with the C-terminus peptide (residues 583–626) demonstrating nucleic acid binding even in the absence of the HD and SAM domains (83, 88). Interestingly, nucleic acid binding by SAMHD1 inhibits dNTPase activity by obstructing the interfaces responsible for tetramer association.…”

“…Interestingly, nucleic acid binding by SAMHD1 inhibits dNTPase activity by obstructing the interfaces responsible for tetramer association. This obstruction can be relieved by increasing the concentration of activating nucleotides (88). SAMHD1 has also been reported to exhibit exonuclease activity on ssRNA and ssDNA (86, 89, 90).…”