The Replisome Organizer (G38P) of Bacillus subtilis Bacteriophage SPP1 Forms Specialized Nucleoprotein Complexes with two Discrete Distant Regions of the SPP1 Genome Riccardo Missich 1 , Frank Weise 1,2 , Sunghee Chai 1 , Rudi Lurz 2 Xiomara Pedre  1,2 and Juan C. Alonso 1 * 1 Campus Universidad Auto Ânoma de Madrid Centro Nacional de Biotecnologõ Âa, CSIC Cantoblanco, 28049 Madrid Spain 2 Max-Planck-Institut fu Èr molekulare Genetik, Ihnestraûe 73, D-14195, Berlin, Germany Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P). G38P speci®cally binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome. One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin (oriL). The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR. Both sites con- tain two types of repeated elements (termed Box AB and A  T-rich region). The K app for the G38P-oriL DNA and G38P-oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively. G38P binds to the dis- tant oriL and oriR sites cooperatively. DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A  T-rich region. Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through pro- tein ± protein interaction. G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it. These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted. # 1997 Academic Press Limited Keywords: phage biology; DNA replication; protein assembly; replication origin; replication initiation protein *Corresponding author Introduction SPP1 is a virulent Bacillus subtilis double- stranded DNA (dsDNA) bacteriophage. The SPP1 genome length is 44.0 kb, but its mature size is 45.5 kb (Tavares et al., 1992). SPP1 DNA is termin- ally redundant (about 3%) and partially circularly permuted (Behrens et al., 1979; Tavares et al., 1992). The terminal redundancy is essential for the vege- tative growth of the phage after DNA injection into a host cell. To generate such a redundancy, SPP1 replication, by a rolling circle mechanism, leads to long concatemers that are encapsidated into a phage prohead by a processive headful mechanism. DNA packaging proceeds unidirec- tionally from right to left (Chai et al., 1995; Figure 1). SPP1 DNA replication begins about two to three minutes post-infection at a ``unique'' origin located about 0.17(0.03) genome length from one of the ends of the phage genome (see Ganesan et al., 1976; McIntosh et al., 1978). These authors, however, disagreed as to the molecular end at which the origin is located. Recently, Pedre  et al. (1994) have reconciled this discrepancy and re- ported that the SPP1-encoded gene 38 and 39 pro- ducts are required for replisome assembly and that G38P binds to two discrete segments that are lo- cated about 0.15(0.03) genome length from the left and right end of the phage genome. An EcoRI digestion of mature phage SPP1 DNA generates 16 DNA fragments, termed EcoRI-1 to EcoRI-16 (Behrens et al., 1979). The left and right binding sites of G38P correspond to DNA segments within Abbreviations used: bp, base-pairs; GXP, the gene x products; HPUra, 6-(p-hydroxyphenylazo)uracil; K app , apparent equilibrium constant; ssDNA, single-stranded DNA ndPAGE non-denaturing PAGE; oriL and oriR, discrete regions to which G38P binds; TCA, trichloro acetic acid. J. Mol. Biol. (1997) 270, 50±64 0022±2836/97/260050±15 $25.00/0/mb971060 # 1997 Academic Press Limited