Molecular Mechanism Underlying ATP-Induced Conformational Changes in the Nucleoprotein Filament of Mycobacterium smegmatis RecA

Abstract

RecA plays a central role in bacterial DNA repair, homologous recombination, and restoration of stalled replication forks by virtue of its active extended nucleoprotein filament. Binding of ATP and its subsequent recognition by the carboxamide group of a highly conserved glutamine (Gln196 in MsRecA) have been implicated in the formation of active RecA nucleoprotein filaments. Although the mechanism of ATP-dependent structural transitions in RecA has been proposed on the basis of low-resolution electron microscopic reconstructions, the precise sequence of events that constitute these transitions is poorly understood. On the basis of biochemical and crystallographic analyses of MsRecA variants carrying mutations in highly conserved Gln196 and Arg198 residues, we propose that the disposition of the interprotomer interface is the structural basis of allosteric activation of RecA. Furthermore, this study accounts for the contributions of several conserved amino acids to ATP hydrolysis and to the transition from collapsed to extended filament forms in Mycobacterium smegmatis RecA (MsRecA). In addition to their role in the inactive compressed state, the study reveals a role for Gln196 and Arg198 along with Phe219 in ATP hydrolysis in the active extended nucleoprotein filament. Finally, our data suggest that the primary, but not secondary, nucleotide binding site in MsRecA isomerizes into the ATP binding site present in the extended nucleoprotein filament.

The second messenger cyclic di‐AMP negatively regulates the expression of Mycobacterium smegmatis recA and attenuates DNA strand exchange through binding to the C‐terminal motif of mycobacterial RecA proteins

“…The crystal structure of a complex of MsRecA with dATP revealed the presence of a second dATP molecule bound to it within the fully ordered C‐terminal domain (Krishna et al, ). However, conformation of the second dATP is different from that of the first dATP molecule; also, the primary (but not the secondary) dATP site in MsRecA isomerizes into the ATP binding pocket in the active nucleoprotein filament (Manjunath et al, ). Based on the data presented here, we infer that the presence of dATP in the MsRecA crystal was incidental and that cyclic di‐AMP is the natural ligand for the second nucleotide binding site embedded in the C‐terminus of MsRecA.…”

“…However, conformation of the second dATP was different from that of the first dATP molecule (which binds at the N-terminal region in the P-loop), suggesting a regulatory role for the nucleotide bound at the second site. Furthermore, the primary (but not the secondary) nucleotide binding site in MsRecA isomerizes into the ATP binding site in the active RecA nucleoprotein filament (Manjunath et al, 2016). The multiple sequence alignment of RecA amino acid sequences of MtRecA and MsRecA with EcRecA reveals significant sequence divergence among these proteins at their C-terminus ( Fig.…”

High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy

“…A homology model for the inactive counterpart of our structure for the active presynaptic RAD51 filament bound to the ATP analogue AMP-PNP (target) was constructed as per our previously reported approach (37) using the MODELLER version 9.14 suite of programmes (38,39). The structure of the E. Coli RecA in the inactive ADP-bound form (PDB: 1XMS, ref.…”

Weaving DNA strands: structural insight on ATP hydrolysis in RecA-induced homologous recombination

“…There is, however, no structural clue about a mechanism for such chemical to rotational energy transfer. The third hypothesis postulates that monomers switch their binding geometries in the filament from ATP- to ADP-favorable interfaces in response to ATP hydrolysis (24,31). They can then exchange the bound ADP for a new ATP and perform reverse interface switching, thus completing the ATP cycle.…”