Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins

Abstract

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of ∼100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact β/α fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by sitedirected mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg 2+ that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.

Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7

“…X-ray crystal structures of the E. coli DnaG primase show that the conserved residues create a central crevice (12,13). These studies also confirm that motifs IV, V, and VI, implicated in the coordination of NTPs and divalent metal cations, form a TOPRIM fold found in topoisomerase (14). Positioned next to the metal binding site is a basic and hydrophobic depression, proposed to interact with the backbone of the template (12,13).…”

Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis1

“…1) contains a tyrosine that is present in all functional homologs and, when mutated in budding yeast, results in the loss of DSB activity [6]. A second region (blocks B^D) shares homology with the Toprim domain common to topoisomerase and DNA primase enzymes, which may participate in cleavage and/or rejoining reactions by coordinating Mg 2 ions [23]. A third region (block E) with higher than average homology is located near the C-terminal end of SPO11, but is not appreciably similar to any previously described motif, and its function is presently unknown.…”

Origins of the machinery of recombination and sex

“…Clear support for this comes from a remarkable relationship between several key DNA handling enzymes; several enzymes that interact with doublestranded DNA to catalyse apparently distinct reactions share a 100-amino acid domain called the Toprim domain because it is found in most topoisomerases and bacterial DnaG primases (Aravind et al, 1998). The Toprim domain carries the active centre that cuts and joins the DNA in topoisomerases of both the Ia and II classes; in DnaG primase this domain is flanked by longer DNA regions: an upstream one with a zinc finger domain and a downstream one with a domain for interaction with DnaB, a DNA helicase protein that helps load it onto the lagging strand.…”

“…Other kinds of proteins with Toprim domains are less universal and may be less ancient. The scattered distribution of OLD endonucleases with Toprim domains, often virally encoded, and of various phage proteins with such domains suggests that they may have arisen secondarily and been distributed by lateral gene transfer after the cenancestor (Aravind et al, 1998).…”

“…Thus the restriction endonuclease NaeI can be converted into a topoisomerase II (unrelated to the natural ones) by a single amino acid substitution (Huai et al, 2000). The fact that early eukaryotes evolved a DNA topoisomerase I entirely unrelated in sequence or 3D structure (hence called Ib) to topoisomerases Ia and II also indicates that it is mechanistically relatively easy to evolve a topoisomerase from other enzymes: topoisomerase Ib is related to the site-specific integrase (sometimes called recombinase) of phage (Aravind et al, 1998). As it is found also in eukaryotic viruses like vaccinia it was probably secondarily acquired by an early eukaryote from an infecting virus -a nice example of lateral gene transfer.…”