The physical and genetic evidence supporting dimerization of DNA polymerase III fits nicely with a structural model for replication. The missing NTD and the exonuclease domain (Exo) in PolC structure are represented correspondingly by a pair of ellipses (purple) and a filled circle (brown). We show here that of the three subunits in the core polymerase… The DNA polymerase III holoenzyme complex contains at least 10 different subunits organized into 3 functionally essential subassemblies: the Pol III core, the beta sliding clamp processivity factor and the clamp-loading complex. for the template for α compared with αε or a decrease in affinity of one of the protein-protein interactions required to establish Each dot represents a single sequence. We also surveyed the combinations of PolIIIα homologs found in genomes in an attempt to get more clues about functional properties of different polymerase groups and a deeper insight into the evolution of bacterial replication systems. A possible explanation of the DnaE2 avoidance in PolC + DnaE3 genomes is that it might be disadvantageous for bacterium to have two related SOS-inducible polymerases. MutH, which functions as a monomer and belongs to a family of type-II restriction endonucleases, incises the newly synthesized strand at a nearby hemi-methylated 5′-GATC-3′ site. Data for all genomes (A and C) and genomes that do not encode PolC (B and D) are depicted separately. Since CTD, similarly to NTD of PolC, is poorly conserved, at least a number of CTDs may have escaped identification. The surfaces are colored according to ConSurf results: variable—cyan, conserved—maroon. DnaE1 polymerases in Bacteroidetes and Fusobacteria also display a significant variability in the PHP metal binding positions. The motif is part of the region, predicted to be intrinsically unstructured (Supplementary Figure S9B). The survey (Figure 3 and Supplementary Table S2) revealed that each polymerase group has a typical architecture. replication system and replication forks on the chromosome of E. coli. The β-clamp binding motif in PolC polymerases is close to the ideal consensus, which is also one of the most potent β-clamp binding variants (76,78). The analysis of surface residue conservation was performed using the ConSurf (60) server supplied with locally constructed multiple sequence alignments for each of the analyzed groups. It has been associated with a novel Zn2+-dependent proofreading exonuclease activity in at least some C-family polymerases (11,72). Bacterial replicative DNA polymerases interact with the DNA sliding clamp to achieve high speed and processivity. E. coli MMR requires activities of 11 proteins/complexes: MutS, MutL, MutH, UvrD (DNA helicase II), four single-stranded specific exonucleases, single-stranded DNA binding protein (SSB), DNA polymerase III holoenzyme, and DNA ligase (Table 1). Replication forks formed with only α are processive. DnaE3 of Thermotogae and DnaE2 of Bacteroidetes have distinctly shorter core sequence regions than other polymerases in corresponding groups. Protein A creates a covalent ester linkage between a tyrosine residue and the 5′-phosphate group of adenylic acid at position 4306 of viral (+)-strand. Thus, we considered that we might not observe any differences between replication forks in the presence The availability of the holoenzyme subunits in purified form has allowed us to investigate their roles at the replication fork. One of the minor variants features domain expansion (duplicated OB domain) in the otherwise canonical architecture. Such a complex travels on ssDNA following a 5′- to 3′-direction, with the concomitant synthesis of short RNA molecules by DnaG to prime DNA synthesis by host DNA polymerase III holoenzyme. The analysis of coevolution patterns of DnaE and PolC polymerases (82) supports the ancient DnaE hypothesis. Therefore, the conservation of the polymerase core (Pol3) is expected. ↵3 M. Olson, J. Carter, H. G. Dallmann, and C. S. McHenry, unpublished data. DNA products were analyzed by electrophoresis through alkaline-agarose gels Significant sequence matches (E-value ≤ 1e–03, at least 40% of query sequence aligned, 10% or higher sequence identity) were retained. Some bacteria and eukaryotes lack adenine methylation and use nicks on the daughter strands as a discrimination mechanism. Low variability. Because these three proteins form such a tight complex, it is expected that they are present at the replication fork. for the next round of primer synthesis (32). until excess positive supercoils accumulate, causing the replication forks to pause, resulting in the formation of an ERI Thus, SeqA plays the role of a negative regulator of initiation in E. coli. Except for the smallest genomes, the fraction of bacteria carrying sole DnaE1 decreases as genomes become larger. Standard rolling circle replication reactions containing the indicated concentrations of primase and HE reconstituted with Distributions for (A) full-length sequences and (B) only the core region [PHP, Pol3 and (HhH)2 domains; residues 6–889 of E. coli DnaE1]. Annotated complete bacterial genomes (Supplementary Table S1) were obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. The label was Phage protein A nicks between (+)-strand nucleotides 4305 and 4306 at the replication origin (30 bp long), releasing the superhelicity of the DNA molecule to give replicative form II (RFII) DNA molecules. In such case, it might be expected that DnaE1 polymerases with the nonfunctional PHP metal binding site would maintain a conserved ε-binding site, while those with the functional PHP active site would not. The preprimosome is constituted by proteins PriA, PriB, PriC, DnaT, and DnaB. Spontaneous suppressor mutations of the slow growth phenotype that have been mapped to α arise very rapidly in these strains.