scholarly journals The Plasmid RK2 Replication Initiator Protein (TrfA) Binds to the Sliding Clamp β Subunit of DNA Polymerase III: Implication for the Toxicity of a Peptide Derived from the Amino-Terminal Portion of 33-Kilodalton TrfA

2006 ◽  
Vol 188 (15) ◽  
pp. 5501-5509 ◽  
Author(s):  
Kritaya Kongsuwan ◽  
Peter Josh ◽  
Marc J. Picault ◽  
Gene Wijffels ◽  
Brian Dalrymple
Keyword(s):  
Specific Interaction ◽  
Replication Initiation ◽  
Stable Complex ◽  
Binding Motif ◽  
Broad Host Range ◽  
Initiator Protein ◽  
Amino Terminal ◽  
Sliding Clamp ◽  
Wide Range ◽  
Plasmid Rk2

ABSTRACT The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli β sliding clamp (β2). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with β2 and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and β2 and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1 (P. D. Kim, T. M. Rosche, and W. Firshein, Plasmid 43:214-222, 2000). The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the β-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent overinitiation in E. coli through its interaction with the initiator protein, DnaA, and β2. Our results support a model in which T1 toxicity is caused by T1 binding to β2, especially when T1 is overexpressed, preventing β2 from interacting with host replication proteins such as Hda during the early events of chromosome replication.

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

2021 ◽  
Vol 49 (6) ◽  
pp. 3394-3408
Author(s):  
Katarzyna Wegrzyn ◽  
Elzbieta Zabrocka ◽  
Katarzyna Bury ◽  
Bartlomiej Tomiczek ◽  
Milosz Wieczor ◽  
...  
Keyword(s):  
Protein Complex ◽  
Structure Prediction ◽  
Essential Feature ◽  
Specific Interaction ◽  
Replication Initiation ◽  
Broad Host Range ◽  
Winged Helix ◽  
Rep Protein ◽  
Mutant Proteins ◽  
Replication Initiator

Abstract 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.

scholarly journals Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

2003 ◽  
Vol 185 (6) ◽  
pp. 1817-1824 ◽  
Author(s):  
Peter D. Kim ◽  
Trevor Banack ◽  
Daniel M. Lerman ◽  
Jeremiah C. Tracy ◽  
Johanna Eltz Camara ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Soluble Fraction ◽  
Host Protein ◽  
Broad Host Range ◽  
Terminal Portion ◽  
Inner Membrane ◽  
E Coli ◽  
Amino Terminal ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

ABSTRACT The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).

scholarly journals Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

2004 ◽  
Vol 186 (11) ◽  
pp. 3508-3515 ◽  
Author(s):  
Mareike Kurz ◽  
Brian Dalrymple ◽  
Gene Wijffels ◽  
Kritaya Kongsuwan
Keyword(s):  
Amino Acid ◽  
Consensus Sequence ◽  
Nitrilotriacetic Acid ◽  
Replication Initiation ◽  
Binding Motif ◽  
Β Subunit ◽  
Related Protein ◽  
Sliding Clamp ◽  
Hydrolysis Of

ABSTRACT In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

2019 ◽  
Vol 16 (3) ◽  
pp. 272-277 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Anders Løbner-Olesen
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Cyclic Peptide ◽  
Replication Initiation ◽  
False Positive Result ◽  
Over Expression ◽  
Initiator Protein ◽  
Dna Replication Initiation ◽  
Cellular Processes ◽  
Initiation Of Dna Replication

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

scholarly journals Putative Cooperative ATP–DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly

2021 ◽  
Vol 22 (12) ◽  
pp. 6643
Author(s):  
Pawel Jaworski ◽  
Dorota Zyla-Uklejewicz ◽  
Malgorzata Nowaczyk-Cieszewska ◽  
Rafal Donczew ◽  
Thorsten Mielke ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Replication Initiation ◽  
Dna Unwinding ◽  
Rich Region ◽  
Initiator Protein ◽  
New Class ◽  
H Pylori ◽  
General Architecture ◽  
Dnaa Box

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

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