title: Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus.
abstract: RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair.
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Upton
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK School of Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors.forename: Jane I
authors.initials: JI
authors.lastname: Grove
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
authors.forename: Akeel A
authors.initials: AA
authors.lastname: Mahdi
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Briggs
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
authors.forename: David S
authors.initials: DS
authors.lastname: Milner
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK School of Health Sciences and Social Care, Division of Biosciences, Brunel University, Uxbridge, London UB8 3PH, UK.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Rudolph
authors.affiliation: Centre for Genetics and Genomics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK bob.lloyd@nottingham.ac.uk.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005798:Genes, Bacterial; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli; C071446:RecG protein, E coli