Interaction with the carboxy-terminal tip of SSB is critical for RecG function in E. coli

Abstract

In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities

“…Abrogation of the SSB interaction leads to an intermediate phenotype in ΔrecG cells during induced dsDNA breaks or DNA gap formation stress, indicating that the SSB interaction is important for the overall activity of RadD. RecG also physically interacts with SSB, forming a complex that is important for RecG cellular activities ( 52 , 53 ). Thus, RecG and/or RecG:SSB complexes may compensate for the absence of the RadD:SSB complex in ΔradD and SSB-binding radD mutant cells.…”