Mutant DNA polymerase delta from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen.
We have isolated and characterized DNA polymerase delta (pol delta) from two thermosensitive Schizosaccharomyces pombe strains, poldeltats1 and poldeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 degreesC poldeltats1 and poldeltats3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol delta. Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol delta. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.
Perderiset
M
M
CNRS-IFC1, Institut de Recherche sur le Cancer, UPR 9044, 7 Rue Guy Moquet BP8, 94801 Villejuif, France.
Maga
G
G
Piard
K
K
Francesconi
S
S
Tratner
I
I
Hübscher
U
U
Baldacci
G
G
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017124:Conserved Sequence; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006358:Hot Temperature; D007700:Kinetics; D018809:Proliferating Cell Nuclear Antigen / Q000494:pharmacology*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013698:Templates, Genetic; D013816:Thermodynamics
D018809:Proliferating Cell Nuclear Antigen; D004258:DNA Polymerase III