Structural basis of Escherichia coli single-stranded DNA-binding protein stimulation of exonuclease I.
Bacterial single-stranded DNA (ssDNA)-binding proteins (SSBs) play essential protective roles in genome biology by shielding ssDNA from damage and preventing spurious DNA annealing. Far from being inert, ssDNA/SSB complexes are dynamic DNA processing centers where many different enzymes gain access to genomic substrates by exploiting direct interactions with SSB. In all cases examined to date, the C terminus of SSB (SSB-Ct) forms the docking site for heterologous proteins. We describe the 2.7-A-resolution crystal structure of a complex formed between a peptide comprising the SSB-Ct element and exonuclease I (ExoI) from Escherichia coli. Two SSB-Ct peptides bind to adjacent sites on ExoI. Mutagenesis studies indicate that one of these sites is important for association with the SSB-Ct peptide in solution and for SSB stimulation of ExoI activity, whereas the second has no discernable function. These studies identify a correlation between the stability of the ExoI/SSB-Ct complex and SSB-stimulation of ExoI activity. Furthermore, mutations within SSB's C terminus produce variants that fail to stimulate ExoI activity, whereas the SSB-Ct peptide alone has no effect. Together, our findings indicate that SSB stimulates ExoI by recruiting the enzyme to its substrate and provide a structural paradigm for understanding SSB's organizational role in genome maintenance.
Lu
Duo
D
Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 550 Medical Science Center, Madison, WI 53706-1532, USA.
Keck
James L
JL
D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D012996:Solutions
D004268:DNA-Binding Proteins; D012996:Solutions; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I