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

Location of mutants in Cdc1 protein. Sequence alignment of the fission yeast Cdc1 protein and its human orthologue p50 in single-letter code. Residues identical between the two proteins are highlighted in yellow. The sites of insertions and point mutations in Cdc1 are indicated by colour-coded triangles (pentapeptide insertions, E and J mutants) and circles (point mutations, A mutants). Grey circles/triangles indicate fully-functional mutants; green circles/triangles indicate partially-functional mutants; magenta circles/triangles indicate non-functional mutants. Note that the A9 and A10 mutants see two adjacent amino acids mutated and are therefore indicated by side-by-side (magenta) circles. Where the same insertion was found more than once, only one allele appears on the figure (see Table 1 for further information). The ten conserved regions defined in the previous study [36] are underlined and indicated by Roman numerals (I – X). The red, cyan and grey bars correspond to positions of α-helices, β-strands and disordered regions in human p50 [10]. The amino acid residues in the p50 protein that are involved in interactions with p66N are shown in green type. See text for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Location of mutants in Cdc1 protein. Sequence alignment of the fission yeast Cdc1 protein and its human orthologue p50 in single-letter code. Residues identical between the two proteins are highlighted in yellow. The sites of insertions and point mutations in Cdc1 are indicated by colour-coded triangles (pentapeptide insertions, E and J mutants) and circles (point mutations, A mutants). Grey circles/triangles indicate fully-functional mutants; green circles/triangles indicate partially-functional mutants; magenta circles/triangles indicate non-functional mutants. Note that the A9 and A10 mutants see two adjacent amino acids mutated and are therefore indicated by side-by-side (magenta) circles. Where the same insertion was found more than once, only one allele appears on the figure (see Table 1 for further information). The ten conserved regions defined in the previous study [36] are underlined and indicated by Roman numerals (I – X). The red, cyan and grey bars correspond to positions of α-helices, β-strands and disordered regions in human p50 [10]. The amino acid residues in the p50 protein that are involved in interactions with p66N are shown in green type. See text for details.

Mentions: In total, 31 mutant alleles were generated by the PSM method (Table 1, Figure 3). Despite their independent origins, DNA sequence analysis revealed that these corresponded to only 20 different mutant alleles (Table 1), indicating that insertion of Tn4430 is not entirely random, as noted previously [30]. The insertions also clustered towards the 5' end of the cdc1+ gene, with 26 of the 31 insertions mapping within the first 420 bp of the 1386 bp ORF. Clustering towards the 5' end of the ORF was observed in a similar study of the S.pombe rfc2+ gene cloned into pBR322 [35]. Thirty of the mutant alleles contained a 15 nt insertion when compared with the wild-type cdc1+ sequence. The remaining allele (Cdc1-E2) contained a 16 nt insertion, presumably the result of an inexact transposition event. This insertion leads to the Cdc1 protein being truncated after amino acid 26. Another insertion also led to production of a truncated protein: Cdc1-J17 was truncated after amino acid 27. Two of the insertions resulted in different insertions at the same amino acid position in the protein (Cdc1-J6 and Cdc1-E9, between amino acids 124 and 125).


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)

Location of mutants in Cdc1 protein. Sequence alignment of the fission yeast Cdc1 protein and its human orthologue p50 in single-letter code. Residues identical between the two proteins are highlighted in yellow. The sites of insertions and point mutations in Cdc1 are indicated by colour-coded triangles (pentapeptide insertions, E and J mutants) and circles (point mutations, A mutants). Grey circles/triangles indicate fully-functional mutants; green circles/triangles indicate partially-functional mutants; magenta circles/triangles indicate non-functional mutants. Note that the A9 and A10 mutants see two adjacent amino acids mutated and are therefore indicated by side-by-side (magenta) circles. Where the same insertion was found more than once, only one allele appears on the figure (see Table 1 for further information). The ten conserved regions defined in the previous study [36] are underlined and indicated by Roman numerals (I – X). The red, cyan and grey bars correspond to positions of α-helices, β-strands and disordered regions in human p50 [10]. The amino acid residues in the p50 protein that are involved in interactions with p66N are shown in green type. See text for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Location of mutants in Cdc1 protein. Sequence alignment of the fission yeast Cdc1 protein and its human orthologue p50 in single-letter code. Residues identical between the two proteins are highlighted in yellow. The sites of insertions and point mutations in Cdc1 are indicated by colour-coded triangles (pentapeptide insertions, E and J mutants) and circles (point mutations, A mutants). Grey circles/triangles indicate fully-functional mutants; green circles/triangles indicate partially-functional mutants; magenta circles/triangles indicate non-functional mutants. Note that the A9 and A10 mutants see two adjacent amino acids mutated and are therefore indicated by side-by-side (magenta) circles. Where the same insertion was found more than once, only one allele appears on the figure (see Table 1 for further information). The ten conserved regions defined in the previous study [36] are underlined and indicated by Roman numerals (I – X). The red, cyan and grey bars correspond to positions of α-helices, β-strands and disordered regions in human p50 [10]. The amino acid residues in the p50 protein that are involved in interactions with p66N are shown in green type. See text for details.
Mentions: In total, 31 mutant alleles were generated by the PSM method (Table 1, Figure 3). Despite their independent origins, DNA sequence analysis revealed that these corresponded to only 20 different mutant alleles (Table 1), indicating that insertion of Tn4430 is not entirely random, as noted previously [30]. The insertions also clustered towards the 5' end of the cdc1+ gene, with 26 of the 31 insertions mapping within the first 420 bp of the 1386 bp ORF. Clustering towards the 5' end of the ORF was observed in a similar study of the S.pombe rfc2+ gene cloned into pBR322 [35]. Thirty of the mutant alleles contained a 15 nt insertion when compared with the wild-type cdc1+ sequence. The remaining allele (Cdc1-E2) contained a 16 nt insertion, presumably the result of an inexact transposition event. This insertion leads to the Cdc1 protein being truncated after amino acid 26. Another insertion also led to production of a truncated protein: Cdc1-J17 was truncated after amino acid 27. Two of the insertions resulted in different insertions at the same amino acid position in the protein (Cdc1-J6 and Cdc1-E9, between amino acids 124 and 125).

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