title: A single residue unique to DinB-like proteins limits formation of the polymerase IV multiprotein complex in Escherichia coli.
abstract: The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.
authors.affiliation: Department of Biology, Northeastern University, Boston, Massachusetts, USA.
authors.forename: Tiziana M
authors.initials: TM
authors.lastname: Cafarelli
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Rands
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Benson
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Rudnicki
authors.forename: Ida
authors.initials: I
authors.lastname: Lin
authors.forename: Veronica G
authors.initials: VG
authors.lastname: Godoy
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019951:DNA Polymerase beta / Q000096:biosynthesis* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D011938:Rec A Recombinases; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli