Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

Abstract

In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 A resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure-function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation.

Physiological and Biochemical Defects in Carboxyl-terminal Mutants of Mitochondrial DNA Helicase

“…increase in DNA-dependent ATPase activity (12), suggesting that the carboxyl-terminal tail of the d-mtDNA helicase may not be required for its function in mtDNA replication. However, we note that a deletion of three carboxyl-terminal residues of the RepA protein abolishes RSF1010 replication while retaining helicase activity in vitro yet does not interfere with replication in the presence of wild type RepA in vivo (26). Because we overexpress the ⌬(597-613) mutant in the presence of endogenous protein, this remains a possibility that warrants further study.…”

“…In several enzymes belonging to the SF4 group of helicases, a deletion or substitution of carboxyl-terminal residues abolishes replication in vivo without affecting enzymatic activities, and these residues were found to be essential for interaction of the helicase with other replication factors (24,26). For example, 7 of the 17 carboxyl-terminal amino acids in T7 gp4 are negatively charged, and the deletion or substitution mutations in these acidic residues results in loss of T7 phage replication (24).…”

“…Furthermore, the extreme carboxyl-terminal region of many SF4 helicases has been shown to be required for interaction with other replication factors. Carboxyl-terminal residues of T7 gp4 interact directly with T7 DNA polymerase and thioredoxin, and the last three carboxyl-terminal residues in the RSF1010 plasmid-encoded RepA helicase are required for replication in vivo (23)(24)(25)(26)(27)(28).…”

Structural Basis of Mechanochemical Coupling in a Hexameric Molecular Motor

“…H3 and H4 Motifs Encompass ␥-Phosphate Sensors-Sensors of the ␥P that are structurally equivalent to Asn-234 in P4 have been identified in hexameric (H3 motif) (26,42,50) as well as SF1 and SF2 helicases (motif III) (2,22,51). A substitution of histidine within the H3 motif of SF4 helicase RepA (His-179) disturbed the coupling between ATP hydrolysis and the concerted binding/release of ssDNA (42).…”

“…A substitution of histidine within the H3 motif of SF4 helicase RepA (His-179) disturbed the coupling between ATP hydrolysis and the concerted binding/release of ssDNA (42). In the monomeric helicase PcrA a mutation of the structurally homologous glutamine decoupled the ATPase activity from DNA unwinding (24).…”