The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit.
DNA polymerase delta (Pol delta) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol delta is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells.
Sanchez Garcia
Javier
J
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
Ciufo
Leonora F
LF
Yang
Xiaowen
X
Kearsey
Stephen E
SE
MacNeill
Stuart A
SA
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics
C090459:CDC1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C491232:POL3 protein, S cerevisiae; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III