Biomedical subjects

Kritaya Kongsuwan

Publications and source records attributed to Kritaya Kongsuwan.

8 recordsLinked to original sources

PubMed2006

Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

Amino Acid Motifs↗

PubMed2006

The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA.

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 beta sliding clamp (beta(2)). 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 beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.

Amino Acid Sequence↗

PubMed2005

Characterization of a new Bacillus thuringiensis endotoxin, Cry47Aa, from strains that are toxic to the Australian sheep blowfly, Lucilia cuprina.

Sixteen isolates of Bacillus thuringiensis, derived from various soil samples collected in Australia, are highly toxic to larvae of the sheep blowfly (Lucilia cuprina). The toxin gene from one of the strains (CAA890) was cloned by genome walking, and sequencing of the cloned fragments revealed a new cry gene, encoding a protein of 1134 amino acid residues, with a theoretical molecular mass of 139,209Da. Based on the amino acid sequence comparison with known Cry delta-endotoxins, the gene was designated cry47Aa. Homology modelling based on known crystal structures of the Cry toxins reveals the differences to be located in the loops of domain II in the putative toxin-receptor binding surfaces between Cry47Aa and the dipteran active Cry2Aa. We also showed that the cry47Aa gene is present in the other isolates that are highly toxic to the sheep blowfly.

Amino Acid Sequence↗

PubMed2005

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina.

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.

Animals↗

PubMed2005

Conservation of eubacterial replicases.

The last 15 years of effort in understanding bacterial DNA replication and repair has identified that the donut shaped beta2 sliding clamp is harnessed by very functionally different DNA polymerases throughout the lifecycle of the bacterial cell. Remarkably, the sites of binding of these polymerases, in most cases, appear to be the same shallow pocket on the beta dimer. In every case, binding of beta2 by the polymerase enhances their processivity of DNA synthesis. This binding site is also the same point of interaction between beta2 and the clamp loader complex, which binds beta2, opens and places it onto the DNA strand and then vacates the site. Beta2 may also be involved in the initiation of DNA replication again via contact through this same site. While much of the research effort has focused on Escherichia coli and Bacillus subtilis, conservation of this complex system is becoming apparent in diverse bacteria.

Bacteria↗

PubMed2004

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins.

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Amino Acid Motifs↗

PubMed2004

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta 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 beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-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 beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta 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.

Adenosine Triphosphatases↗

PubMed2002

Cellular localisation of the clamp protein during DNA replication.

The beta subunit of Escherichia coli DNA polymerase III holoenzyme was fused to the green fluorescent protein GFP. The gene fusion under the control of the heterologous lac promoter was used to replace the wild-type allele in the chromosome. The formation of GFP-beta fluorescent foci in GFP-beta expressing cells required DNA replication and their number per cell was dependent on cell growth. Examination of GFP-beta foci in a synchronous round of replication suggested that DNA replication was accompanied by the recruitment of GFP-beta foci near the midcell, followed by the rapid migration of the foci in opposite directions to the 1/4 and 3/4 positions during DNA replication.

Chromosomes, Bacterial↗