Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

Abstract

DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

Single molecule dynamics at a bacterial replication fork after nutritional downshift

“…We additionally generated functional mVenus fluorescent protein fusions to DnaC and to DnaG, and employed fusions to DnaE and to DnaX, previously shown to be able to functionally replace wild type proteins (43). We found that a fusion of mVenus to the C- Next, we treated cells with concentrations of MMC, HPUra or SHX that led to slowed down growth rate but did not stop growth ( Fig.…”

“…to compare dynamics of replication proteins to those seen after addition of Mitomycin C (MMC), which induces DNA damage supposed to transiently block the progression of forks (11,40,41), or of 6(p-Hydroxyphenylazo)-uracil (HPUra), which reversibly binds to and inhibits DNA polymerase PolC, thereby blocking progression of replication (42).…”

“…In vitro reconstitution of the B. subtilis replisome has demonstrated that PolC is responsible for all leading strand synthesis as well as most lagging strand synthesis, whereas the more error prone and much slower DNA replicase DnaE (25-60 nt/s for DnaE compared to ~500 nt/s for PolC) plays a crucial role in initiating lagging strand synthesis. DnaE is important for extending the lagging strand RNA primer before handing off to PolC, which then completes replication of the Okazaki fragment (11,12). The synergistic relationship between two polymerases in the B. subtilis replisome resembles the synergy found in eukaryotic replication (11,13).…”

“…DnaE is important for extending the lagging strand RNA primer before handing off to PolC, which then completes replication of the Okazaki fragment (11,12). The synergistic relationship between two polymerases in the B. subtilis replisome resembles the synergy found in eukaryotic replication (11,13).…”

“…The other (lagging) strand is synthesized in the opposite direction in short intervals, giving rise to Okazaki fragments (9). The Gram-positive model organism Bacillus subtilis and the rest of the Firmicutes (low-G+C-content Gram-positive bacteria) use two essential DNA polymerases, DnaE and PolC, during replication (10,11). In vivo,…”