The mammalian target of rapamycin (mTOR) downstream of phosphatidylinositol 3-kinase (PI3K) in the growth factor receptor (GFR) pathway is a crucial metabolic sensor that integrates growth factor signals in cells. We recently showed that human papillomavirus (HPV) type 16 exposure activates signaling from GFRs in human keratinocytes. Thus, we predicted that the virus would induce the PI3K/mTOR pathway upon interaction with host cells. We detected activation of Akt and mTOR several minutes following exposure of human keratinocytes to HPV type 16 (HPV16) pseudovirions. Activated mTOR induced phosphorylation of the mTOR complex 1 substrates 4E-BP1 and S6K, which led to induction of the functional protein translational machinery. Blockade of epidermal GFR (EGFR) signaling revealed that each of these events is at least partially dependent upon EGFR activation. Importantly, activation of PI3K/Akt/mTOR signaling inhibited autophagy in the early stages of virus-host cell interaction. Biochemical and genetic approaches revealed critical roles for mTOR activation and autophagy suppression in HPV16 early infection events. In summary, the HPV-host cell interaction stimulates the PI3K/Akt/mTOR pathway and inhibits autophagy, and in combination these events benefit virus infection.

RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3␣, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1⌬. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2⌬. We propose that Sgs1 has helicasedependent functions in replication and helicase-independent functions in DSB repair by HR.The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for maintaining genome stability and cancer suppression. DSBs are produced by ionizing radiation, genotoxic chemicals, and nucleases and when replication forks encounter DNA damage. Broken ends are converted to Rad51 nucleoprotein filaments that search for and invade homologous duplex DNA, producing a Holliday junction (HJ) intermediate. Branch migration of HJs extends or eliminates heteroduplex DNA (hDNA), and mismatches in hDNA are repaired, resulting in a region of localized loss of heterozygosity termed a gene conversion tract.Crossovers accompany some gene conversions, posing risks of deletions, inversions, translocations, and large-scale loss of heterozygosity (31,45,54). The mechanisms that suppress tract lengths and crossovers are important to elucidate because they determine the extent and frequency of the loss of heterozygosity during DSB repair by HR and thereby regulate genome stability.In the yeast Saccharomyces cerevisiae, mitotic and meiotic crossovers are suppressed by Sgs1 (26, 55), a member of the RecQ helicase family that includes five human proteins, three of which (BLM, WRN, and RECQ4) suppress tumors (25). RecQ helicases have conserved structures and interactions with type I topoisomerases (e.g., yeast Top3). Yeast Rmi1 is in complex with Sgs1-Top3 and may promote binding to branched DNAs or Top3 strand passage (10, 43). Yeast sgs1, top3, and rmi1 mutants show DNA damage hypersensitivity, genome instability, slow growth, poor sporulation, and hyperrecombination (10, 43). Sgs1...

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Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or “high-risk” HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.

Chromosomal translocations are common in leukemia, but little is known about their mechanism. Metnase (also termed SETMAR) is a fusion of a histone methylase and transposase protein that arose specifically in primates. Transposases were thought to be extinct in primates because they would mediate deleterious DNA movement. In primates, Metnase interacts with DNA Ligase IV (Lig IV), and promotes non-homologous end joining (NHEJ) DNA repair. We show here that the primate-specific protein Metnase can also enhance NHEJ in murine cells, and can also interact with murine Lig IV, indicating that it integrated into the pre-existing NHEJ pathway after its development in primates. Significantly, expressing Metnase in murine cells significantly reduces chromosomal translocations. We propose that the fusion of the histone methylase SET domain and the transposase domain in the anthropoid lineage to form primate Metnase promotes accurate intra-chromosomal NHEJ, and thereby suppresses inter-chromosomal translocations. Thus, Metnase may have been selected for because it has a function opposing transposases, and may thus play a key role in suppressing translocations that underlie oncogenicity.

The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.

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