DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3-Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31-Pol3 and Pol31-Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

中文翻译：

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稳定的 Pol31-Pol3 界面抵消了 Pol32 消融对修复的不同影响。

DNA 聚合酶 δ 包含催化亚基 Pol3、Pol31 和 Pol32，有助于 DNA 复制和修复。pol31的缺失是致命的，并且损害 Pol3-Pol31 相互作用域会导致对冷、羟基脲 (HU) 和甲基磺酸盐的超敏反应，从而使pol32 Δ 发生表型复制。我们已经确定了pol31中的丙氨酸替代物，它们抑制了pol32 Δ 细胞中的这些缺陷。我们描述了两个突变体pol31-T415A和pol31-W417A，它们映射到介导 Pol31-Pol3 和 Pol31-Rev3 相互作用的溶剂暴露环。该pol31-T415A替代妥协与 Pol3 CysB 域的结合，而 Pol31-W417A 改进了它。重要的是，失去 Pol32，例如pol31-T415A，会导致 Pol3 和 Pol31 蛋白水平降低，而pol31-W417A会恢复这些水平。这些突变对急性 HU 的恢复、断裂诱导的复制和跨病变合成修复途径具有不同的影响。与 HU 上的跨损伤合成和生长不同，pol31-W417A不会恢复pol32 Δ 细胞中断裂诱导复制的丧失，突出了 Pol32 在叉相关修复中的通路特异性作用。有趣的是，HU 上复制叉的 CHIP 分析表明pol32 Δ 和pol31-T415A 间接破坏停滞叉处的 DNA pol α 和 pol ε。