A Central Role for SSB in Escherichia coli RecQ DNA Helicase Function

Robert D Shereda,James L Keck,Douglas A Bernstein

doi:10.1074/jbc.m608011200

Robert D Shereda, James L Keck + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m608011200

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Abstract

RecQ DNA helicases are critical components of DNA replication, recombination, and repair machinery in all eukaryotes and bacteria. Eukaryotic RecQ helicases are known to associate with numerous genome maintenance proteins that modulate their cellular functions, but there is little information regarding protein complexes involving the prototypical bacterial RecQ proteins. Here we use an affinity purification scheme to identify three heterologous proteins that associate with Escherichia coli RecQ: SSB (single-stranded DNA-binding protein), exonuclease I, and RecJ exonuclease. The RecQ-SSB interaction is direct and is mediated by the RecQ winged helix subdomain and the C terminus of SSB. Interaction with SSB has important functional consequences for RecQ. SSB stimulates RecQ-mediated DNA unwinding, whereas deletion of the C-terminal RecQ-binding site from SSB produces a variant that blocks RecQ DNA binding and unwinding activities, suggesting that RecQ recognizes both the SSB C terminus and DNA in SSB.DNA nucleoprotein complexes. These findings, together with the noted interactions between human RecQ proteins and Replication Protein A, identify SSB as a broadly conserved RecQ-binding protein. These results also provide a simple model that explains RecQ integration into genome maintenance processes in E. coli through its association with SSB.

Highlights

Several DNA metabolic activities, and the mechanisms that integrate them into these pathways are important features of cellular genome maintenance networks
In addition to its roles in the RecF pathway, E. coli RecQ cellular activities include the suppression of illegitimate recombination [22] and SOS DNA damage signaling in response to replication fork stalling [23]
Human WRN protein has been found to interact with over a dozen different DNA replication, recombination, and repair proteins [3]. Some of these interactions are conserved with other RecQ proteins (e.g. replication protein A (RPA), the eukaryotic SSB, binds human WRN (30 –32), BLM [31, 33], and RecQ1 [34] and is likely to bind to RecQ5␤ [35]), 3 The abbreviations used are: ITC, isothermal titration calorimetry; ss, single-strand; ExoI, exonuclease I; DSB, double-strand DNA break; RPA, replication protein A; TAP, tandem affinity purification; WH, winged helix; HRDC, helicase and RNaseD C-terminal; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; wt, wild type; OB, oligonucleotide/oligosaccharide-binding

Summary

A Central Role for SSB in Escherichia coli RecQ DNA Helicase Function*

SSB stimulates RecQ-mediated DNA unwinding, whereas deletion of the C-terminal RecQ-binding site from SSB produces a variant that blocks RecQ DNA binding and unwinding activities, suggesting that RecQ recognizes both the SSB C terminus and DNA in SSB1⁄7DNA nucleoprotein complexes These findings, together with the noted interactions between human RecQ proteins and Replication Protein A, identify SSB as a broadly conserved RecQ-binding protein. These results provide a simple model that explains RecQ integration into genome maintenance processes in E. coli through its association with SSB. Deletion of the RecQ-binding site from E. coli SSB produces a variant that strongly inhibits RecQ-mediated DNA binding and unwinding These results establish SSB as an important feature that helps define RecQ substrates. Identification of the RecQSSB interaction helps explain coordination of RecQ activity with other genome maintenance enzymes through SSB-mediated complexes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION