Perderiset M, Maga G, Piard K, Francesconi S, Tratner I, Hübscher U, Baldacci G
CNRS-IFC1, Institut de Recherche sur le Cancer, UPR 9044, 7 Rue Guy Moquet BP8, 94801 Villejuif, France.
Biochem J. 1998 Nov 1;335 ( Pt 3)(Pt 3):581-8. doi: 10.1042/bj3350581.
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.
我们从两个裂殖酵母温度敏感菌株poldeltats1和poldeltats3中分离并鉴定了DNA聚合酶δ（pol δ），这两个菌株在催化亚基的两个不同进化保守结构域发生了突变。在37℃的限制温度下，poldeltats1和poldeltats3突变菌株在细胞周期的S期停止生长。我们发现，在引物末端水平较低时，增殖细胞核抗原（PCNA）对突变酶的体外刺激低于对野生型pol δ的刺激。突变酶对引物（3'-OH）末端的亲和力和持续合成能力与野生型pol δ没有差异。相比之下，Vmax值低于野生型值。主要的体外缺陷似乎是PCNA对突变酶的刺激减少，导致DNA合成速度降低。此外，ts1 pol δ在37℃时不受低浓度PCNA的刺激，尽管低浓度在25℃时能刺激其活性，这表明在引物末端水平较低时的这种热敏感性可能是其在体内的关键缺陷。因此，ts1和ts3 pol δ突变都位于催化亚基中似乎对其与PCNA有效相互作用直接或间接必需的区域。
