ABSTRACT

RFWD3 is an E3 ubiquitin ligase that plays important roles in DNA damage response and DNA replication. We have previously demonstrated that the stabilization of RFWD3 by PCNA at the replication fork enables ubiquitination of the single-stranded binding protein, RPA and its subsequent degradation for replication progression. Here, we report that RFWD3 associates with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA/LRWD1), components of the pre-replicative complex required for the initiation of DNA replication. Overexpression of ORC/ORCA leads to the stabilization of RFWD3. Interestingly, RFWD3 seems to stabilize ORC/ORCA in cells expressing wild type p53, as the depletion of RFWD3 reduces the levels of ORC/ORCA. Further, the catalytic activity of RFWD3 is required for the stabilization of ORC. Our results indicate that the RFWD3 promotes the stability of ORC, enabling efficient pre-RC assembly.

摘要

RFWD3 是一种 E3 泛素连接酶，在 DNA 损伤反应和 DNA 复制中起重要作用。我们之前已经证明，PCNA 在复制叉处对 RFWD3 的稳定化能够泛素化单链结合蛋白 RPA 及其随后的降解以促进复制进程。在这里，我们报告 RFWD3 与起源识别复合体 (ORC) 和 ORC 相关 (ORCA/LRWD1) 相关联，它们是启动 DNA 复制所需的复制前复合体的组成部分。ORC/ORCA 的过表达导致 RFWD3 的稳定。有趣的是，RFWD3 似乎可以稳定表达野生型 p53 的细胞中的 ORC/ORCA，因为 RFWD3 的消耗降低了 ORC/ORCA 的水平。此外，RFWD3 的催化活性是稳定 ORC 所必需的。