RESUMO
In this work we analyzed protein-protein interactions (PPIs) formed by E. coli replication proteins under three disparate bacterial growth conditions. The chosen conditions corresponded to fast exponential growth, slow exponential growth and growth cessation at the stationary phase. We performed affinity purification coupled with mass spectrometry (AP-MS) of chromosomally expressed proteins (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD, NrdB), tagged with sequential peptide affinity (SPA) tag. Composition of protein complexes was characterized using MaxQuant software. To filter out unspecific interactions, we employed double negative control system and we proposed qualitative and quantitative data analysis strategies that can facilitate hits identification in other AP-MS datasets. Our motivation to undertake this task was still insufficient understanding of molecular mechanisms coupling DNA replication to cellular growth. Previous works suggested that such control mechanisms could involve physical interactions of replication factors with metabolic or cell envelope proteins. However, the dynamic replication protein interaction network (PIN) obtained in this study can be used to characterize links between DNA replication and various cellular processes in other contexts.
