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): TripleTOF 5600, TOF/TOF 5800

ORGANISM(S): Escherichia Coli

SUBMITTER: Paulina Czaplewska

LAB HEAD: Paulina Czaplewska

PROVIDER: PXD032398 | Pride | 2024-01-26

REPOSITORIES: Pride

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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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Project description:Faithful duplication of DNA is essential for the maintenance of genomic stability in all organisms. DNA synthesis proceeds bi-directionally with continuous synthesis of leading strand DNA and discontinuous synthesis of lagging strand DNA. Herein, we describe a method of enriching and Sequencing of Protein-Associated Nascent strand DNA (eSPAN) to detect whether a protein binds the leading- and lagging-strands of DNA replication forks. We show that Pol-epsilon, PCNA, Cdc45, Mcm6 and Mcm10 preferentially associate with leading strands, whereas Pol-alpha, Pol32, Pol-delta, Rfa1 and Rfc1 associate with lagging strands of hydroxyurea (HU)-stalled replication forks. In contrast, PCNA is enriched at lagging strands of normal replication forks in wild type cells and HU-stalled forks in cells lacking Elg1. These studies demonstrate a strategy to reveal proteins at leading and lagging strands of DNA replication forks, and suggest that the unloading of PCNA from lagging strands of HU-stalled replication forks helps maintain genome integrity. We synchronized yeast cells at G1 and released into early S phase in the presence of BrdU, a nucleotide analog that can be incorporated into newly synthesized DNA strand, and hydroxyurea (HU), a ribonucleotide reductase inhibitor. HU has no effect on initiation of DNA replication at early replication origins, but inhibit late replication firing. In addition, replication forks are stalled due to depletion of dNTPs. We then performed chromatin-immunoprecipitation of 12 proteins of interest following a standard procedure. Protein-bound DNAs were then reverse-crosslinked and double strand DNA was denatured. Nascent DNA was enriched by immunoprecipitation using anti-BrdU antibodies. The recovered ssDNA was first marked with ligation to one oligo at 3M-bM-^@M-^Y end before conversion to dsDNA for library preparation and sequencing. In this way, the directionality of ssDNA and therefore strand information of each sequenced DNA were known. The sequencing tag was mapped to both Watson (red) and Crick (blue) strands of the reference genome. In addition to ChIP-eSPAN, we also performed BrdU-IP and single strand DNA sequence (BrdU-ssSeq) and protein ChIP followed by single-strand DNA sequencing (ChIP-ssSeq) for each corresponding ChIP-eSPAN experiment. We also performed Mcm4 and Mcm6 ChIP-seq using cells synchronized at G1 phase of the cell cycle for identification of replication origins in comparison with published dataset. Some protein ChIP-ssSeq and ChIP-eSPAN experiments were repeated and the data fits well each other. Therefore, we did not repeat all protein ChIP-ssSeq and ChIP-eSPAN experiments.

Dataset Information

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

Publications

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

Similar Datasets

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