Limits...
Functional mapping of the fission yeast DNA polymerase delta B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis.

Sanchez Garcia J, Baranovskiy AG, Knatko EV, Gray FC, Tahirov TH, MacNeill SA - BMC Mol. Biol. (2009)

Bottom Line: Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50.The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function.Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK. sanchezgarciajavier@yahoo.com

ABSTRACT

Background: DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol delta comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.

Results: To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.

Conclusion: In the absence of a three-dimensional structure of the entire Pol delta complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

Show MeSH

Related in: MedlinePlus

Dominant negative Cdc1 mutants. cdc1-P13 cells carrying plasmids pREP3X, pREP3XH6BN-Cdc1, pREP3XH6BN-Cdc1-J7 or pREP3XH6BN-Cdc1-A10 were grown up in EMM medium at 28°C before being plated onto EMM plates with (+T, right hand panels) or without 5 μg/ml thiamine (-T, left hand panels) and incubated for 4 days at 28°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2734569&req=5

Figure 5: Dominant negative Cdc1 mutants. cdc1-P13 cells carrying plasmids pREP3X, pREP3XH6BN-Cdc1, pREP3XH6BN-Cdc1-J7 or pREP3XH6BN-Cdc1-A10 were grown up in EMM medium at 28°C before being plated onto EMM plates with (+T, right hand panels) or without 5 μg/ml thiamine (-T, left hand panels) and incubated for 4 days at 28°C.

Mentions: Two of the Cdc1 mutant proteins exerted a strong dominant negative phenotype when expressed at high level in cdc1-P13 cells. Transformed cdc1-P13 cells carrying either pREP3XBN-Cdc1-J7 or -A10 were viable at 28°C only when grown on medium containing thiamine to repress the nmt1 promoter (Figure 5). When transferred to medium lacking thiamine, high-level expression of the mutant proteins led to cell cycle arrest (elongated cell phenotype) and a failure to form colonies. Similar behaviour was observed when these mutant proteins were expressed in cdc1–223 cells at 28°C, although the phenotype was somewhat weaker (data not shown).


Functional mapping of the fission yeast DNA polymerase delta B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis.

Sanchez Garcia J, Baranovskiy AG, Knatko EV, Gray FC, Tahirov TH, MacNeill SA - BMC Mol. Biol. (2009)

Dominant negative Cdc1 mutants. cdc1-P13 cells carrying plasmids pREP3X, pREP3XH6BN-Cdc1, pREP3XH6BN-Cdc1-J7 or pREP3XH6BN-Cdc1-A10 were grown up in EMM medium at 28°C before being plated onto EMM plates with (+T, right hand panels) or without 5 μg/ml thiamine (-T, left hand panels) and incubated for 4 days at 28°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734569&req=5

Figure 5: Dominant negative Cdc1 mutants. cdc1-P13 cells carrying plasmids pREP3X, pREP3XH6BN-Cdc1, pREP3XH6BN-Cdc1-J7 or pREP3XH6BN-Cdc1-A10 were grown up in EMM medium at 28°C before being plated onto EMM plates with (+T, right hand panels) or without 5 μg/ml thiamine (-T, left hand panels) and incubated for 4 days at 28°C.
Mentions: Two of the Cdc1 mutant proteins exerted a strong dominant negative phenotype when expressed at high level in cdc1-P13 cells. Transformed cdc1-P13 cells carrying either pREP3XBN-Cdc1-J7 or -A10 were viable at 28°C only when grown on medium containing thiamine to repress the nmt1 promoter (Figure 5). When transferred to medium lacking thiamine, high-level expression of the mutant proteins led to cell cycle arrest (elongated cell phenotype) and a failure to form colonies. Similar behaviour was observed when these mutant proteins were expressed in cdc1–223 cells at 28°C, although the phenotype was somewhat weaker (data not shown).

Bottom Line: Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50.The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function.Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK. sanchezgarciajavier@yahoo.com

ABSTRACT

Background: DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol delta comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.

Results: To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.

Conclusion: In the absence of a three-dimensional structure of the entire Pol delta complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

Show MeSH
Related in: MedlinePlus