Obesity Induces DNA Damage in Mammary Epithelial Cells Exacerbated by Acrylamide Treatment through CYP2E1-Mediated Oxidative Stress.

Obesity and environmental toxins are risk factors for breast cancer; however, there is limited knowledge on how these risk factors interact to promote breast cancer. Acrylamide, a probable carcinogen and obesogen, is a by-product in foods prevalent in the obesity-inducing Western diet. Acrylamide is metabolized by cytochrome P450 2E1 (CYP2E1) to the genotoxic epoxide, glycidamide, and is associated with an increased risk for breast cancer. To investigate how acrylamide and obesity interact to increase breast cancer risk, female mice were fed a low-fat (LFD) or high-fat diet (HFD) and control water or water supplemented with acrylamide at levels similar to the average daily exposure in humans. While HFD significantly enhanced weight gain in mice, the addition of acrylamide did not significantly alter body weights compared to respective controls. Mammary epithelial cells from obese, acrylamide-treated mice had increased DNA strand breaks and oxidative DNA damage compared to all other groups. In vitro, glycidamide-treated COMMA-D cells showed significantly increased DNA strand breaks, while acrylamide-treated cells demonstrated significantly higher levels of intracellular reactive oxygen species. The knockdown of CYP2E1 rescued the acrylamide-induced oxidative stress. These studies suggest that long-term acrylamide exposure through foods common in the Western diet may enhance DNA damage and the CYP2E1-induced generation of oxidative stress in mammary epithelial cells, potentially enhancing obesity-induced breast cancer risk.

The Causes and Consequences of DNA Damage and Chromosomal Instability Induced by Human Papillomavirus.

High-risk human papillomaviruses (HPVs) are the main cause of cervical, oropharyngeal, and anogenital cancers, which are all treated with definitive chemoradiation therapy when locally advanced. HPV proteins are known to exploit the host DNA damage response to enable viral replication and the epithelial differentiation protocol. This has far-reaching consequences for the host genome, as the DNA damage response is critical for the maintenance of genomic stability. HPV+ cells therefore have increased DNA damage, leading to widespread genomic instability, a hallmark of cancer, which can contribute to tumorigenesis. Following transformation, high-risk HPV oncoproteins induce chromosomal instability, or chromosome missegregation during mitosis, which is associated with a further increase in DNA damage, particularly due to micronuclei and double-strand break formation. Thus, HPV induces significant DNA damage and activation of the DNA damage response in multiple contexts, which likely affects radiation sensitivity and efficacy. Here, we review how HPV activates the DNA damage response, how it induces chromosome missegregation and micronuclei formation, and discuss how these factors may affect radiation response. Understanding how HPV affects the DNA damage response in the context of radiation therapy may help determine potential mechanisms to improve therapeutic response.

Self-reversal facilitates the resolution of HMCES DNA-protein crosslinks in cells.

Abasic sites are common DNA lesions stalling polymerases and threatening genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant processing by 5-hydroxymethyl cytosine, embryonic stem cell (ESC)-specific (HMCES) via a DNA-protein crosslink (DPC) that prevents double-strand breaks. Nevertheless, HMCES-DPCs must be removed to complete DNA repair. Here, we find that DNA polymerase α inhibition generates ssDNA abasic sites and HMCES-DPCs. These DPCs are resolved with a half-life of approximately 1.5 h. HMCES can catalyze its own DPC self-reversal reaction, which is dependent on glutamate 127 and is favored when the ssDNA is converted to duplex DNA. When the self-reversal mechanism is inactivated in cells, HMCES-DPC removal is delayed, cell proliferation is slowed, and cells become hypersensitive to DNA damage agents that increase AP (apurinic/apyrimidinic) site formation. In these circumstances, proteolysis may become an important mechanism of HMCES-DPC resolution. Thus, HMCES-DPC formation followed by self-reversal is an important mechanism for ssDNA AP site management.

Self-reversal facilitates the resolution of HMCES-DNA protein crosslinks in cells.

Abasic sites are common DNA lesions that stall polymerases and threaten genome stability. When located in single-stranded DNA (ssDNA), they are shielded from aberrant processing by HMCES via a DNA-protein crosslink (DPC) that prevents double-strand breaks. Nevertheless, the HMCES-DPC must be removed to complete DNA repair. Here, we found that DNA polymerase α inhibition generates ssDNA abasic sites and HMCES-DPCs. These DPCs are resolved with a half-life of approximately 1.5 hours. Resolution does not require the proteasome or SPRTN protease. Instead, HMCES-DPC self-reversal is important for resolution. Biochemically, self-reversal is favored when the ssDNA is converted to duplex DNA. When the self-reversal mechanism is inactivated, HMCES-DPC removal is delayed, cell proliferation is slowed, and cells become hypersensitive to DNA damage agents that increase AP site formation. Thus, HMCES-DPC formation followed by self-reversal is an important mechanism for ssDNA AP site management.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability.

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance.

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.

High-Risk Human Papillomaviruses and DNA Repair.

Human papillomaviruses (HPVs) are small DNA viruses that infect basal epithelial cells and are the causative agents of cervical, anogenital, as well as oral cancers. High-risk HPVs are responsible for nearly half of all virally induced cancers. Viral replication and amplification are intimately linked to the stratified epithelium differentiation program. The E6 and E7 proteins contribute to the development of cancers in HPV positive individuals by hijacking cellular processes and causing genetic instability. This genetic instability induces a robust DNA damage response and activating both ATM and ATR repair pathways. These pathways are critical for the productive replication of high-risk HPVs, and understanding how they contribute to the viral life cycle can provide important insights into HPV's role in oncogenesis. This review will discuss the role that differentiation and the DNA damage responses play in productive replication of high-risk HPVs as well as in the development of cancer.

HMCES Maintains Replication Fork Progression and Prevents Double-Strand Breaks in Response to APOBEC Deamination and Abasic Site Formation.

5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases.

Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling.

Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP7 and 1,5-IP8 Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.

HMCES Maintains Genome Integrity by Shielding Abasic Sites in Single-Strand DNA.

Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.

Human Papillomaviruses Preferentially Recruit DNA Repair Factors to Viral Genomes for Rapid Repair and Amplification.

High-risk human papillomaviruses (HPVs) activate the ataxia telangiectasia mutated-dependent (ATM) DNA damage response as well as the ataxia telangiectasia mutated-dependent DNA-related (ATR) pathway in the absence of external DNA damaging agents for differentiation-dependent genome amplification. Through the use of comet assays and pulsed-field gel electrophoresis, our studies showed that these pathways are activated in response to DNA breaks induced by the viral proteins E6 and E7 alone and independently of viral replication. The majority of these virally induced DNA breaks are present in cellular DNAs and only minimally in HPV episomes. Treatment of HPV-positive cells with inhibitors of both ATM and ATR leads to the generation of DNA breaks and the fragmentation of viral episomes, indicating that DNA breaks are introduced into HPV genomes. These breaks, however, are rapidly repaired through the preferential recruitment of homologous recombination repair enzymes, such as RAD51 and BRCA1, to viral genomes at the expense of cellular DNAs. When HPV-positive cells are treated with hydroxyurea, this recruitment of RAD51 and BRCA1 to viral genomes is greatly enhanced with little recruitment to damaged cellular DNAs and with retention of the ability of viral genomes to amplify. Overall, our studies demonstrated that human papillomaviruses induce breaks into cellular and viral DNAs and that the preferential repair of these lesions in viral episomes leads to genome amplification.IMPORTANCE High-risk human papillomaviruses (HPVs) are the etiologic agents of cervical cancer and are linked to the development of many other anogenital and oropharyngeal cancers. Replication of high-risk HPVs requires the activation of the ataxia telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) DNA repair pathways. Our studies have shown that HPVs activate these pathways by inducing double-strand breaks primarily in cellular DNAs and minimally in viral genomes. Breaks are induced in viral genomes but are rapidly repaired through the preferential recruitment of homologous repair factors such as RAD51 and BRCA1 to HPV episomes. The preferential repair of breaks in viral genomes leads to amplification. Our study identified a novel mechanism by which human papillomaviruses manipulate DNA repair pathways to productively replicate viral genomes. The induction of genetic instability in cellular DNAs likely contributes to the generation of mutations that lead to the development of cancers.

Human papillomaviruses activate and recruit SMC1 cohesin proteins for the differentiation-dependent life cycle through association with CTCF insulators.

Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses.

p38MAPK and MK2 pathways are important for the differentiation-dependent human papillomavirus life cycle.

Amplification of human papillomaviruses (HPV) is dependent on the ATM DNA damage pathway. In cells with impaired p53 activity, DNA damage repair requires the activation of p38MAPK along with MAPKAP kinase 2 (MK2). In HPV-positive cells, phosphorylation of p38 and MK2 proteins was induced along with relocalization to the cytoplasm. Treatment with MK2 or p38 inhibitors blocked HPV genome amplification, identifying the p38/MK2 pathway as a key regulator of the HPV life cycle.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation.

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation.

Human papillomaviruses recruit cellular DNA repair and homologous recombination factors to viral replication centers.

Human papillomaviruses (HPV) activate the ataxia telangiectasia mutated (ATM)-dependent DNA damage response to induce viral genome amplification upon epithelial differentiation. Our studies show that along with members of the ATM pathway, HPV proteins also localize factors involved in homologous DNA recombination to distinct nuclear foci that contain HPV genomes and cellular replication factors. These studies indicate that HPV activates the ATM pathway to recruit repair factors to viral genomes and allow for efficient replication.

Suppression of STAT-1 expression by human papillomaviruses is necessary for differentiation-dependent genome amplification and plasmid maintenance.

High-risk human papillomaviruses (HPVs) infect stratified epithelia to establish persistent infections that maintain low-copy-number episomes in infected basal cells. Amplification of viral genomes occurs upon keratinocyte differentiation, followed by virion synthesis. During persistent HPV infections, viral proteins act to evade surveillance by both innate and adaptive immune responses. One of the primary pathways regulating the innate immune response is the JAK/STAT pathway. Our studies indicate that the expression of STAT-1, but not other members of interferon (IFN)-stimulated gene factor 3 (ISGF-3) complex such as STAT-2 and IFN regulatory factor 9 (IRF9), is selectively suppressed by HPV proteins at the level of transcription. Both E6 and E7 oncoproteins independently suppress the expression of STAT-1, and mutational analyses indicate that the E6 targeting E6-associated protein (E6AP) is responsible for suppression. The levels of STAT-1 proteins increase upon differentiation of both normal and HPV-positive cells but are still significantly reduced in the latter cells. Transient restoration of STAT-1 levels in HPV-positive cells using recombinant retroviruses significantly impaired viral amplification upon differentiation while long-term increases abrogated maintenance of episomes. Similarly, increased levels of STAT-1 induced by gamma interferon treatment inhibited HPV genome amplification upon differentiation. Overall, our findings demonstrate that suppression of STAT-1 expression by HPV proteins is necessary for genome amplification and maintenance of episomes, suggesting an important role for this activity in viral pathogenesis.

The E7 open reading frame acts in cis and in trans to mediate differentiation-dependent activities in the human papillomavirus type 16 life cycle.

Human papillomaviruses (HPVs) are the causative agents of several important genital and other mucosal cancers. The HPV16 E7 gene encodes a viral oncogene that is necessary for the continued growth of cancer cells, but its role in the normal, differentiation-dependent life cycle of the virus is not fully understood. The function of E7 in the viral life cycle was examined using a series of mutations of E7 created in the context of the complete HPV16 genome. The effect of these E7 mutations on key events of the viral life cycle, including immortalization, episomal maintenance, late promoter activation, and infectious virion synthesis, was examined. Our studies show that the pRb binding domain is indispensable for early viral activities, whereas the C-terminal zinc finger domain contributed primarily to very late events. Mutations of the casein kinase II phosphorylation site caused a complex phenotype involving both the function of E7 protein and a cis element necessary for the activation of the late promoter, identifying for the first time a promoter element important for late promoter function in the context of the viral genome. All mutant genomes tested showed reduced viral titers following growth in organotypic raft cultures. These studies clarify the role of E7 as a regulator of late events in the differentiation-dependent HPV life cycle.

Human papillomavirus E7 enhances hypoxia-inducible factor 1-mediated transcription by inhibiting binding of histone deacetylases.

Infection by human papillomaviruses (HPV) leads to the formation of benign lesions, warts, and in some cases, cervical cancer. The formation of these lesions is dependent upon increased expression of proangiogenic factors. Angiogenesis is linked to tissue hypoxia through the activity of the oxygen-sensitive hypoxia-inducible factor 1α (HIF-1α). Our studies indicate that the HPV E7 protein enhances HIF-1 transcriptional activity whereas E6 functions to counteract the repressive effects of p53. Both high- and low-risk HPV E7 proteins were found to bind to HIF-1α through a domain located in the N-terminus. Importantly, the ability of E7 to enhance HIF-1 activity mapped to the C-terminus and correlated with the displacement of the histone deacetylases HDAC1, HDAC4, and HDAC7 from HIF-1α by E7. Our findings describe a novel role of the E7 oncoprotein in activating the function of a key transcription factor mediating hypoxic responses by blocking the binding of HDACs.