Dataset Information

For origin melting Rep nucleoprotein complex accommodates both dsDNA and ssDNA of DUE

ABSTRACT: The replication initiation proteins interact dsDNA located at replication origin and ssDNA of DNA unwinding element (DUE), formed as a result of the destabilization of the double-stranded helix of AT-rich origin region. It is a critical step in the DNA replication initiation; however, the structure of nucleoprotein complex involving initiator protein, dsDNA and/or ssDNA is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), structural and bioinformatic analysis, we defined amino acid residues in plasmid Rep proteins, TrfA and RepE, that are essential for interaction with ssDNA. The study of Rep mutant proteins containing single amino acid substitutions affecting DNA interaction reveals the importance of Rep-ssDNA complexes formation for a dsDNA melting at DUE. Furthermore, the crystal structures obtained for complex of RepE protein with DUE ssDNA, and, RepE complexed with both DUE ssDNA and dsDNA containing RepE specific binding site (iteron) revealed that the plasmid initiator can not only bind iterons and ssDNA DUE separately but also can form a tripartite nucleoprotein complex bringing together specific sequences of replication origin. The presented data strongly supports the loop-back model in which a replication initiator molecule interacts with dsDNA and ssDNA.

INSTRUMENT(S): 5800 TOF/TOF, TripleTOF 5600

ORGANISM(S): Escherichia Coli

SUBMITTER: Paulina Czaplewska

LAB HEAD: Paulina Czaplewska

PROVIDER: PXD032398 | Pride | 2024-01-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DUEoriS14T_a_-_I14_PMC10_matrix_DHB_ref_pos_pr_1_1.mzxml	Mzxml
	DUEoriS14T_a_-_I14_PMC10_matrix_DHB_ref_pos_pr_1_1.pdf	Pdf
	DUEoriS14T_d_-_J13_PMC10_matrix_DHB_ref_pos_pr_1_4.mzxml	Mzxml
	DUEoriS14T_d_-_J13_PMC10_matrix_DHB_ref_pos_pr_1_4.pdf	Pdf
	DUEoriS14T_d_1406_MSMS__-_D3_MSMS_PMC11_1406_sample_1_4.mzxml	Mzxml

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Publications

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

Wegrzyn Katarzyna K Oliwa Monika M Nowacka Marzena M Zabrocka Elżbieta E Bury Katarzyna K Purzycki Piotr P Czaplewska Paulina P Pipka Justyna J Giraldo Rafael R Konieczny Igor I

Nucleic acids research 20231001 19

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 essen ...[more]

PMID: 37713613

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