RT Journal Article
SR Electronic
T1 Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.04.05.486416
DO 10.1101/2022.04.05.486416
A1 Benjamin Orris
A1 Kevin W. Huynh
A1 Mark Ammirati
A1 Seungil Han
A1 Ben Bolaños
A1 Jason Carmody
A1 Matthew D. Petroski
A1 Benedikt Bosbach
A1 David J. Shields
A1 James T. Stivers
YR 2022
UL http://biorxiv.org/content/early/2022/04/06/2022.04.05.486416.abstract
AB SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), enabling the enzyme to localize to stalled replication forks (RFs) and promote their restart. Since localization of a potent dNTPase at stalled RFs is not harmonious with DNA replication, we used a series of kinetic and thermodynamic measurements to explore a hypothesis where the combined effects of T592 phosphorylation and ssDNA binding serves as a dual switch to turn-off SAMHD1 dNTPase activity. We report that phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1. However, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (~15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial baseline dynamics in the C-terminal domain (which contains T592) which may be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.Competing Interest StatementThe authors have declared no competing interest.