Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha

Abstract

Bacteriophage P4 DNA replication depends upon the phage-encoded ␣ protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [␣cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp␣ that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of ␣ protein in vitro. The ␣cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp␣ domain that binds specifically to DNA and that gp␣cr mutations impair this interaction. We hypothesize that gp␣-Cnr interaction is essential for the control of P4 DNA replication.

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

“…The ␣ protein plays four roles: it acts as a replicative helicase, a primase, and an origin recognition protein (37,42) and interacts with the P4 Cnr protein (copy number regulation (34,38). Therefore, the RepA proteins may exert additional activities to the proposed ones that do not become readily obvious by sequence comparisons.…”

“…The P4 ␣ protein has origin binding ability (39) and interacts with the copy number regulation protein Cnr (34,38). The ␣ gene has been reported to contain the sequence for a secondary replication origin for P4 prophage replication (35).…”

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain

“…Furthermore, these residues specify the domain required for the oligomerization of ␣ molecules, since the large complex structures of gp␣⌬4.3-th bridging ori and crr were also observed with ␣ wild-type protein (31). In addition, this domain is also involved in interacting with the P4 Cnr protein (30), which regulates the P4 copy number (28).…”

“…This protein has primase and helicase activities, binds DNA specifically, and interacts with the phage-encoded protein Cnr (copy number regulation) (30,33). Defined domains indicate the modular structure of the ␣ protein (30, 32).…”

“…This protein has primase and helicase activities, binds DNA specifically, and interacts with the phage-encoded protein Cnr (copy number regulation) (30,33). Defined domains indicate the modular structure of the ␣ protein (30,32). The primase domain of gene product ␣ (gp␣) is related in sequence to that of primases of conjugative plasmids (27), and one of the essential motifs is present in each of the known prokaryotic priming enzymes (20,31).…”