A 'gram-negative-type' DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2).
The Streptomyces coelicolor dnaE gene, encoding the catalytic alpha-subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature-sensitive DNA replication mutant, S. coelicolor ts-38. The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli-type pol III alpha-subunit, rather than to the PolC-type alpha-subunit that is known to be essential for replication in the 'low G + C' Gram-positive bacteria such as Bacillus subtilis. The dnaE gene is able to restore replication to a 'slow stop' mutant (ts-38) and a 'fast stop' mutant (ts-114); the dnaE gene of ts-38 carries a single amino acid substitution (Glu-802 to Lys), and the mutation in ts-114 has been mapped between codons 697 and 1062 of dnaE. Mutant ts-38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts-114 is defective in chain elongation. This study provides the first evidence that a DnaE-type pol III is essential for replication in a Gram-positive bacterium. In addition, the complementation studies suggest that the C-terminal 117 residues are not essential for DnaE function in S. coelicolor. When integrated at a distant site on the chromosome, a fragment containing the 3' half of dnaE(codons 697-1179) is capable of rescuing ts-38 (but not ts-114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.
Department of Biomolecular Sciences, UMIST, Manchester, UK.
F
F
Flett
D
D
de Mello Jungmann-Campello
V
V
Mersinias
S L
SL
Koh
R
R
Godden
C P
CP
Smith
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000235:genetics / Q000502:physiology*; D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000502:physiology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics*; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D013936:Thymidine