In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.

a b s t r a c tCdk1 plays an important role in undifferentiated ES cells, but the underlying mechanism remains unclear. This study explores how Cdk1 collaborates with Oct4 to inhibit differentiation in mouse ES cells. We show a direct interaction between Cdk1 and Oct4, whereas other Cdk members, including Cdk2 and Cdk4, fail to associate with Oct4. By immunocytochemistry we show that Cdk1 and Oct4 co-localize in ES cells. The biological function of the Cdk1-Oct4 complex was also addressed. We found that Cdk1 enhances the binding of Oct4 on the trophectoderm marker Cdx2 and promotes Cdx2 repression. This regulation is independent of Cyclins and of the kinase activity of Cdk1. Our study explains how Cdk1 and Oct4 interplay to inhibit ES cell differentiation into trophectoderm and thereby maintain stemness. Structured summary of protein interactions:Cdk1 physically interacts with Oct4 by anti tag coimmunoprecipitation (View interaction) Oct4 binds to SOX-2 by pull down (View interaction) Cdk1 physically interacts with Oct4 and cyclin-B1 by anti bait coimmunoprecipitation (View interaction) Oct4 binds to Cdk1 by pull down (View interaction) Cdk1 and Oct4 colocalize by fluorescence microscopy (View interaction) Oct4 physically interacts with Sox2 by anti bait coimmunoprecipitation (View interaction) Crown

Rev1, a Y‐family DNA polymerase, is involved in the tolerance of DNA damage by translesion DNA synthesis (TLS). Previous studies have shown that the C‐terminal domain (CTD) and ubiquitin (Ub)‐binding (UBM) domains of Rev1 play important roles in UV‐damage tolerance, but how these domains contribute to the process remains unclear. In this study, we created Ub mutations in a proliferating cell nuclear antigen (PCNA)‐Ub fusion that differentially affect its interaction with Rev1 and Polη and found that UV‐damage tolerance depends on its interaction with Rev1 but not Polη. We also created Rev1‐UBM mutations altering its interaction with a PCNA‐Ub fusion and Rev1‐CTD mutations affecting its interaction with Polη and the Rev7 subunit of Polζ. We thus demonstrated that elevated expression of Rev1 alone is sufficient to confer enhanced UV‐damage tolerance and that this tolerance depends on its physical interaction with monoubiquitinated PCNA and Polζ but is independent of Polη. Collectively, these studies reveal central roles played by Rev1 in coordinating UV‐damage response pathway choice in mammalian cells.

Helicobacter pylori strains that deliver the oncoprotein CagA into gastric epithelial cells are the major etiologic agents of upper gastric diseases including gastric cancer. CagA promotes gastric carcinogenesis through interactions with multiple host proteins. Here, we show that CagA also disrupts Wnt-dependent planar cell polarity (Wnt/PCP), which orients cells within the plane of an epithelium and coordinates collective cell behaviors such as convergent extension to enable epithelial elongation during development. Ectopic expression of CagA in Xenopus laevis embryos impaired gastrulation, neural tube formation, and axis elongation, processes driven by convergent extension movements that depend on the Wnt/PCP pathway. Mice specifically expressing CagA in the stomach epithelium had longer pyloric glands and mislocalization of the tetraspanin proteins VANGL1 and VANGL2 (VANGL1/2), which are critical components of Wnt/PCP signaling. The increased pyloric gland length was due to hyperproliferation of cells at the gland base, where Lgr5 + stem and progenitor cells reside, and was associated with fewer differentiated enteroendocrine cells. In cultured human gastric epithelial cells, the N terminus of CagA interacted with the C-terminal cytoplasmic tails of VANGL1/2, which impaired Wnt/PCP signaling by inducing the mislocalization of VANGL1/2 from the plasma membrane to the cytoplasm. Thus, CagA may contribute to the development of gastric cancer by subverting a Wnt/PCP-dependent mechanism that restrains pyloric gland stem cell proliferation and promotes enteroendocrine differentiation.