Structure and function of primase RepB′ encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode

Abstract

For the initiation of DNA replication, dsDNA is unwound by helicases. Primases then recognize specific sequences on the template DNA strands and synthesize complementary oligonucleotide primers that are elongated by DNA polymerases in leading-and lagging-strand mode. The bacterial plasmid RSF1010 provides a model for the initiation of DNA replication, because it encodes the smallest known primase RepB (35.9 kDa), features only 1 single-stranded primase initiation site on each strand (ssiA and ssiB, each 40 nt long with 5 -and 3 -terminal 6 and 13 single-stranded nucleotides, respectively, and nucleotides 7-27 forming a hairpin), and is replicated exclusively in leading strand mode. We present the crystal structure of full-length dumbbell-shaped RepB consisting of an N-terminal catalytic domain separated by a long ␣-helix and tether from the C-terminal helixbundle domain and the structure of the catalytic domain in a specific complex with the 6 5 -terminal single-stranded nucleotides and the C7-G27 base pair of ssiA, its single-stranded 3 -terminus being deleted. The catalytic domains of RepB and the archaeal/eukaryotic family of Pri-type primases share a common fold with conserved catalytic amino acids, but RepB lacks the zinc-binding motif typical of the Pri-type primases. According to complementation studies the catalytic domain shows primase activity only in the presence of the helix-bundle domain. Primases that are highly homologous to RepB are encoded by broad-host-range IncQ and IncQ-like plasmids that share primase initiation sites ssiA and ssiB and high sequence identity with RSF1010.Pri-type ͉ primase DNA complex ͉ single-strand initiator DNA ͉ crystal structure ͉ oriV A t the initiation of DNA replication, the double helix is unwound by helicases, and then primases synthesize complementary oligonucleotide primers on the single-stranded template DNA. These primers are extended by DNA polymerase, continuously at the leading strand and discontinuously at the oppositely-oriented lagging strand, where Okazaki fragments are synthesized. Primase activity depends on specific ssDNA priming sequences that are periodically required for the initiation of the Okazaki fragments on the lagging strand (that are connected by ligase) but only 1 primer is used for the leading strand (1).The broad-host-range IncQ plasmid RSF1010 is maintained in Ͼ30 Gram-negative and 2 Gram-positive bacteria (2, 3). The replication of IncQ and IncQ-like plasmids and their conjugative transfer was studied in Escherichia coli and in vitro. RSF1010 encodes helicase RepA, primase RepBЈ, and replication initiator protein RepC (4, 5) (Fig. S1 A). Additionally, the replication of RSF1010 requires bacterial host replication proteins such as DNA polymerase III and single-stranded binding protein.Primase RepBЈ is activated on 2 specific, 40-nt-long singlestranded segments termed initiators A and B (ssiA and ssiB) with highly similar nucleotide sequences (6, 7). They are arranged on opposite DNA strands as a palindrome in the origin of vegetative re...

Crystal Structure of the Human Primase

“…assists p49 from another molecule) and that two primase molecules are associated at least during dinucleotide synthesis. There is a precedent of that mechanism; the helical domain of prokaryotic primase Rep␤Ј is flexibly tethered to the catalytic domain of the same polypeptide and plays the critical role in synthesis initiation, like p58 C (46). Consistent with this model, it was recently shown that the Ctf4 trimer that couples Prim-Pol ␣ to the eukaryotic replisome can bind two Pol ␣ molecules (47).…”

Insight into the Human DNA Primase Interaction with Template-Primer

“…According to the data in the literature and our results, we can propose that DNA primases from other species, including phages, bacteria, and archaea, bind DNA/RNA in a similar way, where the flexibly tethered accessory domain participates in binding of the template and the initiating NTP (37)(38)(39)(40)(41)(42). Depending on the organism studied, the functional counterpart of p58 C may be located on the catalytic or a different subunit, may contain an [FeS]-cluster or zinc or none of them, and may work in cis or trans orientation.…”

Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

“…The structural data demonstrate that the RNA priming activity of primase is encoded within a unique protein fold that is unrelated to that of other DNA or RNA polymerases. The polymerase fold of archaeal/ eukaryotic PriS has also been identified in an archaeal replicase with combined primase and polymerase activity (17), a bacterial plasmid primase (18), and the polymerase component of bacterial DNA repair ligase D (19). Conservation of aspartic residues in the PriS sequence and site-directed mutagenesis studies indicate that primase uses metal ion-dependent catalysis for nucleotide polymerization (20).…”