Domain structure of phage P4 alpha protein deduced by mutational analysis

Abstract

Bacteriophage P4 DNA replication depends on the product of the ␣ gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the ␣ gene. Sequence analysis of the entire gene predicted a domain structure for the ␣ polypeptide chain (777 amino acid residues, M r 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the ␣ polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for ␣ mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.Bacteriophage P4 is a temperate satellite phage of Escherichia coli that relies on morphogenetic and lysis functions of the helper phage P2 for its propagation (30). The P4 prophage state is maintained either by integration into the host chromosome or by autonomous replication as a multicopy plasmid. Lysogenization of the host, DNA replication during the lytic cycle, and establishment of the plasmid state all occur independently of helper phage (30). Replication of P4 DNA requires only the P4 ␣ gene product (gp␣) and two cis-acting elements, namely, the origin of replication ori and the sequence-related region crr (cis replication region) (7,26,30,63). Another P4 gene, cnr (copy number regulation), maps directly upstream of ␣; its product has been suggested to control P4 DNA replication, in particular at the plasmid level, maybe by direct interaction of Cnr with gp␣ (59). Thus, protein-protein interaction might modulate at least one specific function of the ␣ protein.The ␣ protein is a multifunctional phage replication protein that recognizes ori and crr by binding to octamer sequences (type I repeats, TGTTCACC [16]) (64). In addition, gp␣ catalyzes the unwinding of DNA via its helicase activity. DNA is unwound with a 3Ј to 5Ј polarity with respect to the strand that gp␣ has bound. The helicase action is fueled by nucleoside triphosphate (NTP) hydrolysis of an interrelated singlestranded DNA-dependent NTPase. gp␣ also functions as a primase, with a template-dependent oligonucleotide-synthesizing activity (56, 64). The primase activity of gp␣ can be replaced by the primase of the host, the DnaG protein (56). However, the P4 burst sizes in the presence of a primase-null ␣ protein and an active DnaG protein were always small, indicating that DnaG can only partially substitute for the primase fu...

Identification of Residues in Yeast Spo11p Critical for Meiotic DNA Double-Strand Break Formation

“…For the Glu-449 residue in yeast topoisomerase II (equivalent to Spo11p Glu-233), replacement by alanine completely abolished DNA cleavage activity, similar to the case for Spo11p and consistent with the fact that topoisomerase II requires a divalent metal cofactor for DNA cleavage (29). Similarly, a glutamine substitution for Glu-214 in bacteriophage P4 ␣ protein abolished primase activity in vitro and in vivo (46). In contrast, mutations at Glu-9 of E. coli topoisomerase I behaved somewhat differently.…”

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

“…Looping between ori and crr was detected in vitro in the presence of gp␣ in supercoiled or linear P4 DNA (29). The DNA binding activity of ␣ protein lies near its C terminus (30), while the primase activity is near the N terminus (26). Both domains containing these activities can function independently from each other: the N-terminal truncation containing the primase domain complements a P4 primase-null phage and retains primase activity in vitro (26).…”

“…The C-terminal 150 amino acids of gp␣ have specific DNA binding activity in vitro. The helicase domain, probably spanning the middle and the C-terminal third of gp␣, requires the integrity of a large fraction of the protein (28).…”

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

“…Hence, in all our studies we made use of the advantages this genetically well-characterized host bacterium (15) served as host for the pSH plasmids. Media were as described previously (41). When appropriate, antibiotics were added to the following concentrations: ampicillin (sodium salt; 100 g/ml), chloramphenicol (10 g/ml), tetracycline · HCl (10 g/ml), or neomycin (30 g/ml).…”