The oncoprotein E7 from human papillomavirus-16 (HPV-16 E7) plays a pivotal role in HPV postinfective carcinogenesis, and its physical interaction with host cell targets is essential to its activity. We identified a novel cellular partner for the viral oncoprotein: the actin-binding protein gelsolin (GSN), a key regulator of actin filament assembly and disassembly. In fact, biochemical analyses, generation of a 3D molecular interaction model and the use of specific HPV-16 E7 mutants provided clear cut evidence supporting the crucial role of HPV-16 E7 in affecting GSN integrity and function in human immortalized keratinocytes. Accordingly, functional analyses clearly suggested that stable HPV-16 E7 expression induced an imbalance between polymeric and monomeric actin in favor of the former. These events also lead to changes of cell cycle (increased S phase), to the inhibition of apoptosis and to the increase of cell survival. These results provide support to the hypotheses generated from the 3D molecular interaction model and encourage the design of small molecules hindering HPV-induced host cell reprogramming by specifically targeting HPV-16 E7-expressing cells.
The viral factor E7 plays a key role in the well-established association between "high-risk" Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano-genital cancer. To delve into the molecular mechanisms of HPV-mediated cell transformation, we searched for novel potential cellular targets of the HPV-16 E7 oncoprotein, by means of the yeast two-hybrid technique, identifying a protein-protein interaction between HPV-16 E7 and the pro-apoptotic cellular factor Siva-1. Using co-precipitation assays and the "PepSets" technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV-16 E7 competed in vitro with the binding of the Bcl-X(L) anti-apoptotic factor to Siva-1, an interaction that has a major inference in UV radiation-induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV-16 E7 expression by retroviral infection caused Siva-1 transcript up-regulation, detected by cDNA macroarray hybridization and real-time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti-apoptotic role of HPV-16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva-1 expression produced a significant increase of the apoptotic rate in UV radiation-exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV-16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti-apoptotic role of HPV-16 E7, performed at least by its interference with Siva-1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones.
Abstract Understanding how transcriptional programs help to coordinate cell growth and division is an important unresolved problem. Here we report that the nutrient-and stress-regulated transcription factor Sfp1 is rate-limiting for expression of several large classes of genes involved in yeast cell growth, including ribosomal protein, ribosome biogenesis, and snoRNA genes. Remarkably, the spectrum of Sfp1 transcription effects is concordant with a combination of chromatin immunoprecipitation and chromatin endogenous cleavage binding analyses, which together provide evidence for two distinct modes of Sfp1 promoter binding, one requiring a co-factor and the other a specific DNA-recognition motif. In addition to growth-related genes, Sfp1 binds to and regulates the promoters of cell cycle “START” regulon genes, including the key G1/S cyclins CLN1 and CLN2 . Our findings suggest that Sfp1 acts as a master regulator of cell growth and cell size by coordinating the expression of genes implicated in mass accumulation and cell division.
The DNA Damage Tolerance pathway (DDT) is one of the major mechanisms for resolving replication fork blocks. A central factor for DDT is the fork-associated clamp, PCNA, which can undergo to mono- or polyubiquitination, leading to error-free or error-prone modes of repair, respectively. The Rad5 protein in the yeast Saccharomyces cerevisiae plays important roles in both pathways: promoting the error-free mode by PCNA polyubiquitination and interacting with specialized DNA polymerases involved in the error-prone pathway. Rad5 also associates with telomeres, the repetitive DNA regions present at the ends of chromosomes. Telomeric DNA, tightly bound by tandem proteins arrays, poses unique challenges to replication fork progression. Here, I review the current understanding of the link between Rad5 and telomeres and present evidence that Rad5 is associated with yeast telomeres, with notable enrichment during telomere replication. This data highlights a connection between telomeres and a key DDT factor in unperturbed wild-type cells, raising intriguing possibilities about the functional interplay between telomere replication and DNA damage tolerance mechanisms.
BackgroundDNA replication in eukaryotes is initiated from specific chromosomal sites (origins) that fire in a defined, cell type-specific temporal pattern.This replication program appears to be under epigenetic control through mechanisms that are still poorly understood.
The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.
Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, e.g., Rap1 in Saccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Ku's NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJ in vivo without disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexes in vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Ku's inward translocation on DNA - an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Ku's inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.
Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells.
