Critical Review Protein – Protein Interactions in the Eubacterial Replisome Patrick M. Schaeffer, Madeleine J. Headlam and Nicholas E. Dixon Research School of Chemistry, Australian National University, Canberra ACT 0020, Australia Summary Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7. IUBMB Life, 57: 5–12, 2005 Keywords DNA replication; Escherichia coli; Bacillus subtilis; bacteriophage; initiation; elongation; termination. INTRODUCTION Replication of DNA in all organisms proceeds in three stages: initiation at origins of replication, DNA synthesis (elongation) at replication forks, and termination. Each of these processes is mediated by multiple stable or transient protein – protein and protein – DNA interactions involving subsets of the 30 or so different replication proteins (Fig. 1). The elongation phase occurs within a large nucleoprotein assembly called the replisome (1). Replisomes have never been isolated intact from cells, so understanding of their structures and functions has required their painstaking in vitro reassem- bly from individual subunits. Mechanistic studies have made extensive use of the Escherichia coli proteins, in part because they are separately isolable in large quantities from over- producing strains, and in vitro initiation, elongation and termination reactions have been faithfully reconstituted using defined DNA templates. Other systems studied in similar detail include the replication of the DNA of bacteriophages. Some, like T4 and T7 encode their own replication proteins while others like the small single-stranded (ss) DNA phages make use of subsets of host enzymes. Similar reductionist approaches have more recently been applied to identify many of the replication proteins from archaebacteria and eukar- yotes. It is clear that protein functions and mechanisms are conserved in all organisms (Table 1), though the proteins may have little or no recognizable sequence similarity. Work in the last ten years has produced a good under- standing of how protein – protein interactions mediate repli- somal DNA synthesis in E. coli (Fig. 2A), and high-resolution structures of (or domains of) many of the individual proteins and some larger complexes are now available. For all of these reasons, it is apparent that E. coli DNA replication provides an excellent opportunity to study the chemistry that governs macromolecular interactions in a flexible and dynamic nucleoprotein assembly. The E. coli replisome can thus be seen as a tractable model system to develop knowledge and tools that may be applied to other large dynamic multiprotein complexes. Such complexes mediate or control many aspects of development and function in organisms from all domains of life. ASSEMBLY OF TWO REPLISOMES AT ORIC How replisomes are assembled is probably the least well understood process in replication. In the 4.7 million base pairs of the circular E. coli chromosome is a 260-bp sequence that constitutes the unique origin of replication (oriC). All bacteria have replication origins that contain multiple copies of short DNA sequences recognized by replication initiator proteins. In Received 11 September 2004; accepted 15 December 2004 Address correspondence to: Dr. Nicholas Dixon, Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia. E-mail: dixon@rsc.anu.edu.au; Tel: + 61 – 2 – 61254391; Fax: + 61 – 2 – 61250750 IUBMB Life, 57(1): 5–12, January 2005 ISSN 1521-6543 print/ISSN 1521-6551 online # 2005 IUBMB DOI: 10.1080/15216540500058956