Abstract
BackgroundDNA polymerase δ 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 δ 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 δ 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.ResultsTo 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.ConclusionIn the absence of a three-dimensional structure of the entire Pol δ 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.
Highlights
DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand
Extending from the RNA-DNA primers laid down by DNA polymerase α (Pol α)-primase, recent results indicate that DNA polymerase ε (Pol ε) is likely to replicate the bulk of the leading strand [2] while DNA polymerase δ (Pol δ) synthesises the bulk of the lagging strand [3], reviewed by [4,5]
Very little high-resolution three-dimensional structural information on the three polymerases was available, as the only solved structures were those of a 38 amino acid zinc finger derived from the carboxy-terminal end of the catalytic subunit of human Pol α and 75 amino acids of the aminoterminal domain of the B-subunit of human Pol ε, both determined by NMR [7,8]
Summary
DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol and three smaller subunits Cdc, Cdc and Cdm. Three evolutionarily-conserved DNA polymerase enzymes play essential roles at the eukaryotic replication fork [1]. DNA polymerase α-primase is essential for the initiation of leading strand synthesis at individual replication origins and for the initiation of each Okazaki fragment on the discontinuously-synthesised lagging strand. Very little high-resolution three-dimensional structural information on the three polymerases was available, as the only solved structures were those of a 38 amino acid zinc finger derived from the carboxy-terminal end of the catalytic subunit of human Pol α and 75 amino acids of the aminoterminal domain of the B-subunit of human Pol ε, both determined by NMR [7,8]. High-resolution structures are available for the complex of p50 (the second subunit) and the N-terminal 144 amino acids of p66 (the third subunit) of human Pol δ (designated p50p66N) [10,11] and for the complex of the C-terminal domain of budding yeast Pol protein, the catalytic subunit of Pol α, and the C-terminal PDE and OB fold domains of the Pol α B-subunit Pol12 [12]
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