A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site

Abstract

The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.Despite its importance, information on the molecular mechanism of termination of replication of the bacterial chromosome has lagged far behind that on the initiation and elongation phases of the replication cycle (7). There is evidence for the existence of sequence-specific terminators in the chromosomes of Bacillus subtilis 168 (11-13) and Escherichia coli (2, 4). In B. subtilis, the termination process appears to be less complex. A single termination site, called terC, is located approximately opposite the origin, oriC. The clockwise-moving replication fork reaches terC first and is arrested. The counterclockwise fork arrives a few (<5) minutes later, presumably to fuse with the arrested fork and to complete termination. Arrest of the clockwise fork at terC represents the first stage in the overall process of termination. The sequence of 1.3 kilobases of DNA spanning terC has been determined (1). Arrest occurs within this sequence in the vicinity of two imperfect inverted repeats (IRs; 47 and 48 nucleotides each, separated by 59 nucleotides) and just upstream of an open reading frame (ORF) encoding a small basic protein (Mr, 14,519). The -200 base pairs (bp) of DNA spanning the IRs is referred to as the IR region (IRR). Experiments with strains in which portions of the sequence on either side of terC were deleted or modified have given information on the sequence requirements for clockwise fork arrest (10). In particular, disruption of the ORF by insertion of four extra nucleotides abolished fork arrest. It was proposed that arrest is dependent upon binding of the ORF-encoded protein to the IRR within which terC is located. In this report, we describe the purification of this protein from B. subtilis and the results of experiments which establish that the protein does bind specifically to the IRR as proposed. Thus, a protein required for termination of replication of a bacterial chromosome, now called the replication terminator (RT) protein, was identified. Figure 1 summarizes the relevant features of the DNA sequence spanning terC in B. subtilis. The clockwise fork enters this sequence from the right and is arrested at terC just upstream of the ORF for the RT protein (designated the rtp gene) and in the vicinity of IRI and IRII, which make up the IRR (1). NdeI cuts this sequence between the ribosomebinding site and the initiation codon of the ORF; the HaeIII site lies approximately 120 nucleotides downstream of the termination codon of the ORF. The 480-bp NdeI-HaeIII portion was inserted, via a number of steps, into the multiple cloning site of E. coli expression vector pKK223-3 (Pharma-* Corresponding au...

Role of the EBNA-1 Protein in Pausing of Replication Forks in the Epstein-Barr Virus Genome

“…In Bacillus subtilis, the replication arrest system consists of an inverted repeat (IR) region containing two inverted repeats (IR I and IR II) and the replication terminator protein (RTP), which associates as a dimer with IR I and IR II. The IR sequences contain core and auxiliary elements, each of which binds an RTP dimer and results in a polar barrier to replication fork movement (47,48). Pausing of replication forks in vitro occurs only when RTP binds to the core and auxiliary elements cooperatively (49 -51).…”

Replication Fork Stalling at Natural Impediments

“…Each Ter site is bipartite, i.e., it consists of two elements, core and auxiliary sites, and each of those elements binds one homodimer of the replication termination protein, RTP (the molecular weight of a monomer is 14.5 kDa). RTP was purified (158) and shown to arrest replication in vitro in an orientation-dependent manner when bound to the TerI (IRI) and TerII (IRII) sites (124,271). RTP and Tus share very little sequence similarity, and the crystal structure of RTP revealed a lack of structural similarity and confirmed that RTP exists as a symmetrical dimer (28).…”

Replication fork arrest at relocated replication terminators on the Bacillus subtilis chromosome

“…Cells were grown in Spizizen medium plus methionine (50 g/ml), thymine (50 g/ml), tryptophan (40 g/ml), and Casamino Acids (0.1%). DNA was extracted as described above and cut with BamHI-EcoRI or EcoRI alone, and 200 ng of each digest per lane was electrophoresed on 0.7% agarose gels in Tris-phosphate-EDTA (14) at 45 V for 18 to 24 h. After being stained with 25 g of ethidium bromide per ml, the DNA was transferred from the gels to nylon membranes with a Bio-Rad Vacuumblotter according to Bio-Rad's instructions. After transfer, the gels were restained to verify the complete transfer of DNA.…”