Clamp Loader Processing Is Important during DNA Replication Stress

Though the presence of DnaX tau and gamma forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. The DNA clamp loader is critical to the processivity of the DNA polymerase and coordinating synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full-length tau form and shorter gamma form. These are conserved across bacterial species, and three distinct mechanisms have been found to create them: ribosomal frameshift, transcriptional slippage, and, in Caulobacter crescentus, partial proteolysis. This conservation suggests that DnaX processing is evolutionarily important, but its role remains unknown. Here we find a bias against switching from expression of a wild-type dnaX to a nonprocessable tau-only allele in Caulobacter. Despite this bias, cells are able to adapt to the tau-only allele with little effect on growth or morphology and only minor defects during DNA damage. Motivated by transposon sequencing, we find that loss of the gene sidA in the tau-only strain slows growth and increases filamentation. Even in the absence of exogenous DNA damage treatment, the Delta sidA tau-only double mutant shows induction of and dependence on recA, likely due to a defect in resolution of DNA damage or replication fork stalling. We find that some of the phenotypes of the Delta sidA tau-only mutant can be complemented by expression of gamma but that an overabundance of tau-only dnaX is also detrimental. The data presented here suggest that DnaX processing is important during resolution of DNA damage events during DNA replication stress. Although the presence of DnaX tau and gamma forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both tau and gamma is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms are important during the resolution of DNA replication stress.IMPORTANCE Though the presence of DnaX tau and gamma forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both tau and gamma is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms is important during the resolution of DNA replication stress.

Automated summary

Different species of bacteria have developed different mechanisms to produce DnaX tau and gamma forms, suggesting the importance of having two forms of DnaX. DnaX is critical for the processivity of DNA polymerase and coordinating synthesis on the leading and lagging strands. Despite being discovered over 30 years ago, the purpose of expressing both tau and gamma forms is still unclear. This study provides evidence that expressing two forms of DnaX and controlling their abundance and/or ratio is important during the resolution of DNA replication stress.