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Affinity purification of an archaeal DNA replication protein network.

Li Z, Santangelo TJ, Cuboňová L, Reeve JN, Kelman Z - MBio (2010)

Bottom Line: Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery.Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research.The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA.

ABSTRACT
Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His(6)-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

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The interaction network documented for protein components of the T. kodakarensis replisome. Proteins that were His6 tagged and used to isolate interacting proteins are identified in colored ovals (the colors used are as in Fig. 1). Proteins that were coisolated with a His6-tagged protein are identified in white ovals by the designations of the T. kodakarensis genes that encode them.
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f2: The interaction network documented for protein components of the T. kodakarensis replisome. Proteins that were His6 tagged and used to isolate interacting proteins are identified in colored ovals (the colors used are as in Fig. 1). Proteins that were coisolated with a His6-tagged protein are identified in white ovals by the designations of the T. kodakarensis genes that encode them.

Mentions: Lysates were generated from exponentially growing but not synchronized cell populations and so contained complexes present at all stages of the replication cycle. The His6-tagged proteins (Table 1) were all synthesized as soluble proteins and were present in readily detectable amounts in the clarified lysates. All of the putative protein-protein interactions detected, based on the coisolation of a protein with a His6-tagged protein, are documented in Table S1 in the supplemental material. The consistent interactions that remained, after the exclusion of proteins whose annotated functions argue strongly against a role in nucleic acid metabolic processes, are listed in Table 2 and illustrated as a network in Fig. 2. Many of the interactions were confirmed by coisolation of the same proteins when different interacting partners were His6 tagged and used to isolate the complex. The results include both previously established and previously unknown interactions between documented, predicted, and previously unrecognized components of the archaeal replisome. In some cases, when two or three homologous proteins with very similar sequences were present and different homologues were His6 tagged, the same proteins were coisolated, consistent with functional redundancy. When this was not the case, the results argue for divergence of the homologues to the extent that different interactions are made, suggesting different functions.


Affinity purification of an archaeal DNA replication protein network.

Li Z, Santangelo TJ, Cuboňová L, Reeve JN, Kelman Z - MBio (2010)

The interaction network documented for protein components of the T. kodakarensis replisome. Proteins that were His6 tagged and used to isolate interacting proteins are identified in colored ovals (the colors used are as in Fig. 1). Proteins that were coisolated with a His6-tagged protein are identified in white ovals by the designations of the T. kodakarensis genes that encode them.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962436&req=5

f2: The interaction network documented for protein components of the T. kodakarensis replisome. Proteins that were His6 tagged and used to isolate interacting proteins are identified in colored ovals (the colors used are as in Fig. 1). Proteins that were coisolated with a His6-tagged protein are identified in white ovals by the designations of the T. kodakarensis genes that encode them.
Mentions: Lysates were generated from exponentially growing but not synchronized cell populations and so contained complexes present at all stages of the replication cycle. The His6-tagged proteins (Table 1) were all synthesized as soluble proteins and were present in readily detectable amounts in the clarified lysates. All of the putative protein-protein interactions detected, based on the coisolation of a protein with a His6-tagged protein, are documented in Table S1 in the supplemental material. The consistent interactions that remained, after the exclusion of proteins whose annotated functions argue strongly against a role in nucleic acid metabolic processes, are listed in Table 2 and illustrated as a network in Fig. 2. Many of the interactions were confirmed by coisolation of the same proteins when different interacting partners were His6 tagged and used to isolate the complex. The results include both previously established and previously unknown interactions between documented, predicted, and previously unrecognized components of the archaeal replisome. In some cases, when two or three homologous proteins with very similar sequences were present and different homologues were His6 tagged, the same proteins were coisolated, consistent with functional redundancy. When this was not the case, the results argue for divergence of the homologues to the extent that different interactions are made, suggesting different functions.

Bottom Line: Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery.Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research.The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA.

ABSTRACT
Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His(6)-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

Show MeSH