Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

15 p.-6 fig.An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00].Peer reviewe

Rep protein accommodates together dsDNA and ssDNA which enables a loop-back mechanism to plasmid DNA replication initiation

17 p.-8 fig.-1 graph. abst.For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the replication origin. The structural–functional relationship of the nucleoprotein complex involving initiator proteins is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), the analysis of mutant proteins and crystal structures, we defined amino acid residues essential for the interaction between plasmid Rep proteins, TrfA and RepE, and ssDNA. This interaction and Rep binding to dsDNA could not be provided in trans, and both are important for dsDNA melting at DNA unwinding element (DUE). We solved two crystal structures of RepE: one in a complex with ssDNA DUE, and another with both ssDNA DUE and dsDNA containing RepE-specific binding sites (iterons). The amino acid residues involved in interaction with ssDNA are located in the WH1 domain in stand β1, helices α1 and α2 and in the WH2 domain in loops preceding strands β1’ and β2’ and in these strands. It is on the opposite side compared to RepE dsDNA-recognition interface. Our data provide evidence for a loop-back mechanism through which the plasmid replication initiator molecule accommodates together dsDNA and ssDNA.Polish National Science Centre grant [2017/26/D/NZ1/00239]; E.Z. was supported by the MPD/2010/5 project operated within the Foundation for Polish Science International PhD Projects (MPD) Programme co-financed by the EU European Regional Development Fund; I.K. was supported by Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00]; R.G. is the recipient of a grant by Spanish MICIN/AEI [PID2021-124866OB-I00]. Funding for open access charge: University of Gdansk.Peer reviewe