DNA polymerase eta (Pol eta) is a member of the mammalian Y family polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol eta accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol eta, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol eta through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol eta into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol eta-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol eta-mediated translesion synthesis.

Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20-hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR-A and usp-2 , but not EcR-B1 or usp-1 , may be components of the ecdysone receptor complex. Up-regulation of E75A, BHR3 , and three BR-C isoforms, but not E75B , appeared to be associated with the induction of PCD. β β β β FTZ-F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.

Heat shock transcription factor 1 (HSF1) regulates the expression of a wide array of genes, controls the expression of heat shock proteins (HSPs) as well as cell growth. Although acute depletion of HSF1 induces cellular senescence, the underlying mechanisms are poorly understood. Here, we report that HSF1 depletion-induced senescence (HDIS) of human diploid fibroblasts (HDFs) was independent of HSP-mediated proteostasis but dependent on activation of the p53-p21 pathway, partly because of the increased expression of dehydrogenase/reductase 2 (DHRS2), a putative MDM2 inhibitor. We observed that HDIS occurred without decreased levels of major HSPs or increased proteotoxic stress in HDFs. Additionally, VER155008, an inhibitor of HSP70 family proteins, increased proteotoxicity and suppressed cell growth but failed to induce senescence. Importantly, we found that activation of the p53-p21 pathway resulting from reduced MDM2-dependent p53 degradation was required for HDIS. Furthermore, we provide evidence that increased DHRS2 expression contributes to p53 stabilization and HDIS. Collectively, our observations uncovered a molecular pathway in which HSF1 depletion-induced DHRS2 expression leads to activation of the MDM2-p53-p21 pathway required for HDIS.

REV1 is a Y-family polymerase that plays a central role in mutagenic translesion DNA synthesis (TLS), contributing to tumor initiation and progression. In a current model, a monoubiquitinated form of the replication accessory protein, proliferating cell nuclear antigen (PCNA), serves as a platform to recruit REV1 to damaged sites on the DNA template. Emerging evidence indicates that posttranslational mechanisms regulate REV1 in yeast; however, the regulation of REV1 in higher eukaryotes is poorly understood. Here we show that the molecular chaperone Hsp90 is a critical regulator of REV1 in human cells. Hsp90 specifically binds REV1 in vivo and in vitro. Treatment with a specific inhibitor of Hsp90 reduces REV1 protein levels in several cell types through proteasomal degradation. This is associated with suppression of UV-induced mutagenesis. Furthermore, Hsp90 inhibition disrupts the interaction between REV1 and monoubiquitinated PCNA and suppresses UV-induced focus formation. These results indicate that Hsp90 promotes folding of REV1 into a stable and/or functional form(s) to bind to monoubiquitinated PCNA. The present findings reveal a novel role of Hsp90 in the regulation of TLS-mediated mutagenesis.Genomic DNA is constantly exposed to both extrinsic and intrinsic genotoxic agents, such as UV light and oxidative stress. Although most DNA lesions are removed by multiple DNA repair pathways, some escape these mechanisms and persist in the genome. Such unrepaired DNA lesions usually block the progression of replication forks catalyzed by highfidelity DNA polymerases (Pols), which may lead to downstream reinitiation of DNA synthesis, leaving single-stranded DNA gaps. Translesion synthesis (TLS) is an essential mechanism for bypassing such replication blocks and postreplicative gaps by employing specialized Pols, including Pol , Pol , Pol , and REV1, members of the Y-family Pols (Y-Pols), and Pol , a member of the B family (10,14,28,43,58). These TLS Pols display low stringency for the active site and a lack of proofreading, and thus they contribute to mutagenesis and to DNA damage tolerance. Recruitment of the TLS Pols at sites of DNA damage depends on Rad6/Rad18-and CRL4Cdt2 -mediated production of monoubiquitinated proliferating cell nuclear antigen (PCNA) (Ub-PCNA), to which Y-Pols bind with high affinity (3, 21, 54, 57).REV1 plays a central role in promoting mutagenesis in lower and higher eukaryotes. Of note, the mutagenic activity of REV1 contributes to tumor initiation and progression in mammals (9, 29, 62). REV1 has a unique deoxycytidyl transferase activity in vitro, incorporating dCMP opposite several types of DNA lesions, such as abasic sites and damaged guanines (17,18,30,38,65); however, this enzymatic activity is dispensable for REV1-mediated TLS (37). Instead, the function of REV1 depends on protein-protein interactions via three key domains: the N-terminal BRCT domain (named after the C-terminal domain of a breast cancer susceptibility protein) (15,22,23), ubiquitin (Ub)-binding motifs (UBMs) in th...

Programmed cell death of larval-specific tissues in insects is under the control of 20-hydroxyecdysone (20E). The ecdysteroid-regulated early genes are conserved in the programmed cell death of anterior silk glands (ASGs) in Bombyx mori and salivary glands in Drosophila melanogaster. We identified and characterized two isoforms of the Ets transcription factor E74 gene in B. mori (BmE74). In ASGs of B. mori last instar larvae, the Bm74A mRNA level increased concomitantly with an increase in haemolymph ecdysteroid titre after gut purge. The optimal 20E concentration for stimulation of Bm74A in ASGs was 4 microM, a similar value to the peak haemolymph ecdysteroid concentration after gut purge. In contrast, BmE74B expression peaked on day 5 of the feeding period, after which it did not increase again. These findings suggest that the BmE74 isoforms play different roles in the regulation of programmed cell death in B. mori ASGs.