The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase g (polg) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that polg does not form nuclear foci in RAD18 À/À cells after UV irradiation. Both Rad18 and Rad6 are required for polg focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with polg constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, polg interacts preferentially with monoubiquitinated PCNA, but pold does not. These results suggest that Rad18 is crucial for recruitment of polg to the damaged site through protein-protein interaction and PCNA monoubiquitination.

Summary Error-free repair of DNA double-strand breaks (DSB) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway1. In the absence of BRCA1-mediated HR, administration of PARP inhibitors induces synthetic lethality of tumor cells of patients with breast or ovarian cancers2,3. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration4. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases5. In particular, little is known about BRCA1-independent restoration of HR. Here, we show that loss of REV7 (also known as MAD2L2) re-establishes CtIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and appears to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance6. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining (NHEJ) during immunoglobulin class switch recombination. Our results reveal an unexpected critical function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Planarians are known for their strong regenerative ability. This ability has been considered to reside in the totipotent somatic stem cell called the "neoblast." Neoblasts contain a unique cytoplasmic structure called the "chromatoid body," which has similar characteristics to the germline granules of germline cells of other animals. The chromatoid bodies decrease in number and size during cytodifferentiation and disappear in completely differentiated cells during regeneration. However, germ cells maintain the chromatoid body during their differentiation from neoblasts. These observations suggest that the chromatoid body is concerned with the totipotency of cells. To understand the molecular nature of the chromatoid body in the neoblast, we focused on vasa (vas)-related genes, since VAS and VAS-related proteins are known to be components of the germline granules in Drosophila and Caenorhabditis elegans. By PCR, two vas-related genes (Dugesia japonica vasa-like gene, DjvlgA and DjvlgB) were isolated, and they were shown to be expressed in germ cells. Interestingly, DjvlgA was also expressed in a number of somatic cells in the mesenchymal space. In regenerating planarians, accumulation of DjvlgA-expressing cells was observed in both the blastema and the blastema-proximal region. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjvlgA-expressing cells decreased drastically. These results suggest that the product of DjvlgA may be a component of the chromatoid body and may be involved in the totipotency of the neoblast.

Planarians are considered to be among the most primitive animals which developed the central nervous system (CNS). To understand the origin and evolution of the CNS, we have isolated a neural marker gene from a planarian, Dugesia japonica, and analyzed the structure of the planarian CNS by in situ hybridization. The planarian CNS is located on the ventral side of the body, and composed of a mass of cephalic ganglions in the head region and a pair of ventral nerve cords (VNC). Cephalic ganglions cluster independently from VNC, are more dorsal than VNC, and form an inverted U-shaped brain-like structure with nine branches on each outer side. Two eyes are located on the dorsal side of the 3(rd) branch and visual axons form optic chiasma on the dorsal-inside region of the inverted U-shaped brain. The 6(th)-9(th) branches cluster more closely and form auricles on the surface which may function as the sensory organ of taste. We found that the gross structure of the planarian CNS along the anterior-posterior (A-P) axis is strikingly similar to the distribution pattern of the "primary" neurons of vertebrate embryos which differentiate at the neural plate stage to provide a fundamental nervous system, although the vertebrate CNS is located on the dorsal side. These data suggest that the basic plan for the CNS development along the A-P axis might have been acquired at an early stage of evolution before conversion of the location of the CNS from the ventral to the dorsal side.

To analyze the organization of planarian brain, a homolog of the homeobox-containing gene Orthopedia (Otp) from planarian, Djotp, was isolated. The homeodomain of Djotp differs from mouse Otp by only two amino acids. This conservation extends to include a 12 amino acid motif downstream of the homeodomain. Whole mount in situ hybridization studies indicated that Djotp is specifically expressed in the branch structures of the normal planarian adult brain. During regeneration, Djotp is expressed in the presumptive branch region prior to branch formation. These observations implicate a role for Djotp in establishing and maintaining the identity of the planarian brain branch region. The results suggest that recruitment of Otp for its role in brain pattern formation occurred very early in evolution.