The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

Abstract

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

DNA Repair and Genome Maintenance in Bacillus subtilis

“…Gene annotations for RecQ and RecS are different depending on the source of information. RecQ is encoded by an open reading frame named either recQ or yocI, whereas RecS is encoded by an open reading frame named either recQ or ypbC, again depending on the source used (77,350). B. subtilis RecQ shares the closest sequence homology to human WRN and E. coli RecQ, as it contains the highly conserved helicase domain, the RecQ helicase conserved region (RecQ-Ct), and the helicase and RNase D C-terminal domains (HRDC) (Fig.…”

“…Recent work showed that the SSB protein targets several proteins, including RecQ, RecS, and RecJ, to the replication fork (77). Proteomic analysis of the interactome of SSB revealed numerous DNA repair proteins that bind SSB in vivo, including but not limited to RecQ, RecS, and RecJ (77) (see Table 3 for a complete list).…”

Defining the Intrinsically Disordered C-Terminal Domain of SSB Reveals DNA-Mediated Compaction

“…The recently postulated maintenance hub theory ascribes a speculative role for SSBs as a scaffold hub that recruits proteins involved in DNA metabolism and localises them to ssDNA [1].…”

“…This is followed by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 C-terminal end of the arm, which binds to SSB interaction partners. Although this acidic region is well conserved in different species, their SSB interactomes are species dependent [1].…”

Role of RPA Proteins in Radiation Repair and Recovery

“…Akin to requirements for ATR activation, multiple RPAs are necessary to promote this exchange. Similarly, bacterial SSB interacts through its C-terminal tail with the recombination mediator RecO to facilitate loading of RecA onto ssDNA by RecFOR (Costes, et al, 2010;Inoue, et al, 2011). Another interesting interaction of SSB is its apparent competition with the RecA inhibitor RecX for the common ssDNA ligand.…”