Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase delta subunit Cdc27.
BACKGROUND
DNA polymerases alpha and delta play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase alpha (Pol alpha)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase delta (Pol delta) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase epsilon (Pol epsilon).


RESULTS
Here it is shown that the catalytic subunit of Pol alpha, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol delta, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function.


CONCLUSIONS
A short protein sequence motif (DPIM) has been identified as mediating Pol alpha-Pol delta interactions in fission yeast. Despite being conserved across species, mutational analysis indicates the DPIM does not play an essential role in vivo, suggesting that interaction between the two polymerases is also non-essential.
Gray
Fiona C
FC
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK. fgray@ed.ac.uk
Pohler
J Richard G
JR
Warbrick
Emma
E
MacNeill
Stuart A
SA
D020816:Amino Acid Motifs / Q000235:genetics; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D020134:Catalytic Domain; D018797:Cell Cycle Proteins; D017124:Conserved Sequence; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III; D000074002:DNA Polymerase gamma; D004261:DNA Replication / Q000502:physiology; D004271:DNA, Fungal / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D020798:Two-Hybrid System Techniques
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577366:CDC27 protein, human; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C513024:cdc27 protein, S pombe; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase