SummaryAn early expressed operon, located at the right end of the linear bacteriophage f29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in f29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a f29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo f29 DNA replication is discussed.

A.Serna-Rico and D.Mun Äoz-Espõ Ân contributed equally to this workRemarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage f29 as a model system. Previously, we demonstrated that the f29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated f29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the f29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo f29 DNA replication is presented. Keywords: Bacillus subtilis/bacteriophage f29/in vivo DNA replication/ssDNA binding/terminal protein interaction IntroductionEukaryotic DNA replication occurs at numerous ®xed positions within the nucleus, as assessed by microscopic imaging techniques, implying that they are attached to subcellular structures (reviewed in Cook, 1999). During recent years, microscopic imaging tools have been developed for prokaryotic research and the results obtained have contributed importantly to a better understanding of in vivo prokaryotic DNA replication and related processes (Jensen and Shapiro, 2000). One of the most important recent contributions is the discovery that replicative DNA polymerase of Bacillus subtilis is located at relatively stationary cellular positions (Lemon and Grossman, 1998). This study had a vast impact on the view of prokaryotic DNA replication. First, it implied that the replicating DNA template moves through the stationary polymerase, contrary to the generally accepted view that the DNA polymerase moves along the DNA during replication. Second, it indicated that DNA polymerase, together with other proteins involved in DNA replication, are organized in so-called stationary replication factories. Finally, the stationary position of the replication factory entails that it is attached to a substructure. This adapted view of DNA replication, which most probably applies to all bacteria, shows remarkable similarities to that of eukaryotic DNA replication (reviewed in Cook, 1999), indicating that the basic principles of prokaryotic and eukaryotic DNA replication are more conserved than was previously thought.Compelling evidence has been provided during the past few decades that prokaryotic DNA replication, including that of resident plasmids and infecting phages, occurs at the cellular membrane (for review see Firshein, 1989), which most probably is the ...

The linear double-stranded genome of phage 29 contains a terminal protein (TP) covalently linked at each 5 DNA end, called parental TP. Initiation of 29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing 29 DNA polymerase and primer TP. It has been argued that origin recognition involves protein-protein interactions between parental and primer TP. Analysis of the TP sequence revealed that the region between amino acids 84 and 118 has a high probability to form an amphipatic ␣-helix that could be involved in the interaction between parental and primer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted amphipatic ␣-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic ␣-helix of TP is an important determinant involved in origin recognition.Before DNA polymerase duplicates a DNA strand using its complementary strand as template, DNA replication has to be initiated. This process involves several distinct steps such as recognition of the origins, unwinding of double-strand (ds) 1 DNA, and priming (for reviews, see Refs. 1 and 2). Genomes consisting of a linear dsDNA molecule with a terminal protein (TP) covalently linked to their 5Ј-ends have been found in bacteriophages (e.g. 29, 15, Nf, B103, GA-1, Cp-1, and PRD1), animal viruses (e.g. adenoviruses), plasmids (e.g. S1 and Kalilo), and bacteria (e.g. Streptomyces). In most of these cases, initiation of replication has been shown to occur via a so-called protein-priming mechanism, which has been studied extensively for the Bacillus subtilis phage 29 (for reviews, see Refs. 3 and 4). . The formation of this first TP-dAMP covalent complex is directed by the second nucleotide at the 3Ј-end of the template; then, the TP-dAMP complex slides back one nucleotide to recover the information of the terminal nucleotide (8). Next, the 29 DNA polymerase synthesizes a short elongation product before dissociating from the TP (9). Replication, which starts at both DNA ends, is coupled to strand displacement. This results in the generation of so-called type I replication intermediates consisting of fulllength double-stranded 29 DNA molecules with one or more single-stranded DNA branches of varying lengths. When the two converging DNA polymerases merge, a type I replication intermediate becomes physically separated into two type II replication intermediates. Each of these consists of a fulllength 29 DNA molecule in which a portion of the DNA, starting from one end, is double-stranded and the portion spanning to the other end is single-stranded (10, 11). Continuous elongation by the DNA polymerase completes replication of the parental strand.The 29 DNA polymerase and TP form a stable heterodimer (12). This complex recognizes the origin of replication, probably through protein-protein interactions betwee...