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Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD
Author(s) -
Murison David Andrew,
Timson Rebecca,
Beuning Penny
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.754.7
Subject(s) - sos response , dna damage , dna , cleave , homologous recombination , chemistry , biology , biochemistry , microbiology and biotechnology , genetics
The SOS response, an induced DNA damage response pathway in Escherichia coli , is temporally separated into two stages. The initial stage affords an opportunity for the cell to utilize accurate repair mechanisms such as homologous recombination and nucleotide excision repair. During the later stage, cells employ a potentially mutagenic pathway termed translesion synthesis (TLS) to tolerate damage and promote survival. The umuD gene products are upregulated during the SOS response and play a role in both early and late stages. Full‐length UmuD is expressed as a homodimer of 139‐amino‐acid subunits which eventually cleave their N‐terminal 24 amino acids to form UmuD′. The cleavage product, UmuD′, together with UmuC, form the Y‐family polymerase DNA Pol V (UmuD′ 2 C) capable of performing TLS. UmuD and UmuD′ exist as homodimers, but their subunits can readily exchange to form UmuDD′ heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway for the pro‐mutagenic UmuD′. Full‐length UmuD is targeted for degradation by the ClpXP protease due to a recognition sequence located on the N‐terminal arm of UmuD. Formation of UmuD′ via cleavage removes this recognition sequence, making UmuD′ invisible to ClpX. Thus, UmuD in the context of heterodimer acts as a delivery factor for UmuD′. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange for a number of single‐cysteine UmuD variants. By conjugating fluorescent molecules at different positions on the protein we were able to observe their effect on exchange kinetics. Interestingly, the most dramatic differential effects on exchange were observed at two adjacent positions in the peptide backbone. These findings provide insight into the molecular surfaces responsible for mediating exchange. Support or Funding Information Supported by NSF CAREER MCB‐0845033 and American Cancer Society RSG‐12‐161‐01‐DMC.