Specific amino acid residues in the β sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli

• Published in: DNA repair 2006-03, Vol.5 (3), p.312-323
• Main Authors: Sutton, Mark D., Duzen, Jill M.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Amino Acids - genetics; Amino Acids - metabolism; Bacteriology; Biological and medical sciences; DNA Polymerase beta - genetics; DNA Polymerase beta - metabolism; DNA Polymerase beta - radiation effects; DNA Polymerase II - genetics; DNA Polymerase II - metabolism; DNA Polymerase II - radiation effects; DNA polymerase selection/switching; DNA Replication; DNA, Bacterial - biosynthesis; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; DNA-Directed DNA Polymerase - radiation effects; dnaN alleles; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Growth, nutrition, cell differenciation; Microbiology; Models, Molecular; Molecular and cellular biology; Molecular genetics; Mutagenesis; Mutagenesis. Repair; Mutation; Phenotype; Translesion DNA synthesis; Ultraviolet Rays; β sliding clamp
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2005.10.011

Escherichia coli dnaN159 strains encode a mutant form of the β sliding clamp (β159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the β clamp with the α catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (β-G174A), while the other contained D173A, G174A and H175A substitutions (β-173–175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Δ polB (pol II), Δ dinB (pol IV) and/or Δ umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the β clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others’ abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in β159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.