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Presents a model for the switching of prokaryotic DNA polymerase complexes with eukaryotic polymerase delta complex during the initiation of DNA replication. SV40 DNA replication in vitro; Proteins required for DNA replication; Substitution by phage DNA polymerase complexes; Bidirectional DNA replication.

Prostate cancer is a significant health concern and the most commonly diagnosed cancer in men worldwide. Understanding the complex process of prostate tumor evolution and progression is crucial for improved diagnosis, treatments, and patient outcomes. Previous studies have focused on unraveling the dynamics of prostate cancer evolution using phylogenetic or lineage analysis approaches. However, those approaches have limitations in capturing the complete disease process or incorporating genomic and transcriptomic variations comprehensively. In this study, we applied a novel computational approach to derive a prostate cancer progression model using multidimensional data from 497 prostate tumor samples and 52 tumor-adjacent normal samples obtained from The Cancer Genome Atlas study. The model was validated using data from an independent cohort of 545 primary tumor samples. By integrating transcriptomic and genomic data, our model provides a comprehensive view of prostate tumor progression, identifies crucial signaling pathways and genetic events, and uncovers distinct transcription signatures associated with disease progression. Our findings have significant implications for cancer research and hold promise for guiding personalized treatment strategies in prostate cancer., Significance: We developed and validated a progression model of prostate cancer using >1,000 tumor and normal tissue samples. The model provided a comprehensive view of prostate tumor evolution and progression., (©2024 The Authors; Published by the American Association for Cancer Research.)

A major function of the DNA damage responses (DDRs) that act during the replicative phase of the cell cycle is to inhibit initiation and elongation of DNA replication. It has been shown that DNA replication of the polyomavirus, SV40, is inhibited and its replication fork is slowed by cellular DDR responses. The inhibition of SV40 DNA replication is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral DNA helicase. Mass spectroscopy was used to identify a novel highly conserved DDR kinase site, T518, on LT. In cell-based assays expression of a phosphomimetic form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication, but LT-dependent transcriptional activation was unaffected. Purified WT and LT T518D were analyzed in vitro. In concordance with the cell-based data, reactions using SV40 LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication. A myriad of LT protein-protein interactions and LT's biochemical functions were unaffected by the LT T518D mutation; however, LT's DNA helicase activity was dramatically decreased on long, but not very short, DNA templates. These results suggest that DDR phosphorylation at T518 inhibits SV40 DNA replication by suppressing LT helicase activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53 Mut ) cancer are largely ineffective. Here, we report a therapeutic strategy for p53 Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53 Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53 Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53 Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53 Mut cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Polyomavirus ( PyV ) Large T-antigen ( LT ) is the major viral regulatory protein that targets numerous cellular factors/pathways: tumor suppressors, cell cycle regulators, transcription and chromatin regulators, as well as other factors for viral replication. LT directly recruits the cellular replication factors involved in LT's recognition of the viral origin, origin unwinding, and primer synthesis which is carried out by mutual interactions between LT, DNA polymerase alpha-primase ( Polprim ), and single strand (ss) DNA binding replication protein A ( RPA ). The activities as well as interactions of these three with each other as well as other factors, are known to be modulated by post-translational modifications (PTMs); however, modern high-sensitivity proteomic analyses of the PTMs as well as proteins associated with the three have been lacking. Elution from immunoprecipitation (IP) of the three factors were subjected to high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors: 82 PAARs on SV40 LT, 305 on the Polprim heterotetrametric complex and 92 on the RPA heterotrimeric complex. LC-MS/MS analysis also identified proteins that co-immunoprecipitated (coIP-ed) with the three factors that were not previously reported: 374 with LT, 453 with Polprim and 183 with RPA. We used a bioinformatic-based approach to analyze the proteomics data and demonstrate a highly significant "enrichment" of transcription-related process associated uniquely with LT, consistent with its role as a transcriptional regulator, as opposed to Polprim and RPA associated proteins which showed no such enrichment. The most significant cell cycle related network was regulated by ETS proto-oncogene 1 (ETS1), indicating its involvement in regulatory control of DNA replication, repair, and metabolism. The interaction between LT and ETS1 is validated and shown to be independent of nucleic acids. One of the novel phosphorylated aa residues detected on LT from this study, has been demonstrated by us to affect DNA replication activities of SV40 Large T-antigen. Our data provide substantial additional novel information on PAARs, and proteins associated with PyV LT, and the cellular Polprim-, RPA- complexes which will benefit research in DNA replication, transformation, transcription, and other viral and host cellular processes.

Background: The identification of cancer driver genes and key molecular pathways has been the focus of large-scale cancer genome studies. Network-based methods detect significantly perturbed subnetworks as putative cancer pathways by incorporating genomics data with the topological information of PPI networks. However, commonly used PPI networks have distinct topological structures, making the results of the same method vary widely when applied to different networks. Furthermore, emerging context-specific PPI networks often have incomplete topological structures, which pose serious challenges for existing subnetwork detection algorithms., Methods: In this paper, we propose a novel method, referred to as MultiFDRnet, to address the above issues. The basic idea is to model a set of PPI networks as a multiplex network to preserve the topological structure of individual networks, while introducing dependencies among them, and, then, to detect significantly perturbed subnetworks on the modeled multiplex network using all the structural information simultaneously., Results: To illustrate the effectiveness of the proposed approach, an extensive benchmark analysis was conducted on both simulated and real cancer data. The experimental results showed that the proposed method is able to detect significantly perturbed subnetworks jointly supported by multiple PPI networks and to identify novel modular structures in context-specific PPI networks.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.

Ewing sarcoma is the second most common bone malignancy in children and adolescents. In recent years, a large body of evidence has emerged that suggests Ewing tumors harbor large amounts of replication stress (RS). CDC7, also known as DDK (DBF4-dependent kinase), is a serine/threonine kinase that is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the RS response. Due to DDK's diverse roles during replication, coupled with the fact that there is an increased level of RS within Ewing tumors, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibition. Here, we report that DDK inhibition resulted a significant reduction in cell viability and the induction of apoptosis, specifically in Ewing sarcoma cells. Treatment with DDK inhibitors dramatically reduced the rate of replication, prolonged S-phase, and led to a pronounced increase in phospho-CDC2 (Y15), indicating delay of mitotic entry. The induction of cell death corresponded to mitotic exit and G1 entry, suggesting improper mitotic progression. In accordance with this, we find that DDK inhibition caused premature mitotic entry resulting in mitotic abnormalities such as anaphase bridges, lagging chromosomes, and cells with >2 poles in Ewing sarcoma cells. This abnormal progression through mitosis resulted in mitotic catastrophe as evidenced by the formation of micronuclei and induction of DNA damage. Together, these findings suggest that DDK activity is required for the faithful and timely completion of DNA replication in Ewing cells and that DDK inhibition may present a viable therapeutic strategy for the treatment of Ewing sarcoma., (© 2022. The Author(s).)

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Breast carcinomas commonly carry mutations in the tumor suppressor p53, although therapeutic efforts to target mutant p53 have previously been unfruitful. Here we report a selective combination therapy strategy for treatment of p53 mutant cancers. Genomic data revealed that p53 mutant cancers exhibit high replication activity and express high levels of the Base-Excision Repair (BER) pathway, whereas experimental testing showed substantial dysregulation in BER. This defect rendered accumulation of DNA damage in p53 mutant cells upon treatment with deoxyuridine analogues. Notably, inhibition of poly (ADP-ribose) polymerase (PARP) greatly enhanced this response, whereas normal cells responded with activation of the p53-p21 axis and cell cycle arrest. Inactivation of either p53 or p21/CDKN1A conferred the p53 mutant phenotype. Preclinical animal studies demonstrated a greater anti-neoplastic efficacy of the drug combination (deoxyuridine analogue and PARP inhibitor) than either drug alone. This work illustrates a selective combination therapy strategy for p53 mutant cancers that will improve survival rates and outcomes for thousands of breast cancer patients., (© 2021. The Author(s).)

Ewing sarcoma is an aggressive pediatric tumor of the bone and soft tissue. The current standard of care is radiation and chemotherapy, and patients generally lack targeted therapies. One of the defining molecular features of this tumor type is the presence of significantly elevated levels of replication stress as compared with both normal cells and many other types of cancers, but the source of this stress is poorly understood. Tumors that harbor elevated levels of replication stress rely on the replication stress and DNA damage response pathways to retain viability. Understanding the source of the replication stress in Ewing sarcoma may reveal novel therapeutic targets. Ewing sarcomagenesis is complex, and in this review, we discuss the current state of our knowledge regarding elevated replication stress and the DNA damage response in Ewing sarcoma, one contributor to the disease process. We will also describe how these pathways are being successfully targeted therapeutically in other tumor types, and discuss possible novel, evidence-based therapeutic interventions in Ewing sarcoma. We hope that this consolidation will spark investigations that uncover new therapeutic targets and lead to the development of better treatment options for patients with Ewing sarcoma. IMPLICATIONS: This review uncovers new therapeutic targets in Ewing sarcoma and highlights replication stress as an exploitable vulnerability across multiple cancers., (©2020 American Association for Cancer Research.)

The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication.

The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ's replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1's helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BK virus nephropathy is a challenging clinical problem in kidney transplant recipients with wide range of surveillance and management practices, based on individual experience. BK virus reactivation in kidney transplant recipients can result in BK virus nephropathy and graft loss. The most effective strategy for early diagnosis and treatment of BK virus nephropathy is regular monitoring for BK virus, currently achieved by quantification of viral DNA in blood by quantitative polymerase chain reaction. Immunosuppression reduction remains the mainstay of treatment; however, viral clearance is often followed by acute rejection, likely secondary to a delay between immune reconstitution and viral clearance. Impaired cell-mediated immune response to BK virus has been shown to correlate with progression to BK virus nephropathy, while reconstitution of this response correlates with resolution of nephropathy. There is recent research to support monitoring BK virus-specific cell-mediated immune response as a predictor of disease progression and resolution. In this article, we review the current concepts and recent developments in understanding BK virus-associated disease in the context of kidney transplant and outline areas for future research.

Unlabelled: The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner., Importance: While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner., (© 2013 Elsevier Inc. All rights reserved.)

Human papillomavirus (HPV) infections are a major human health problem; they are the cause of recurrent benign warts and of several cancers of the anogenital tract and head and neck region. Although there are two prophylactic HPV vaccines that could, if used universally, prevent as many as two-thirds of HPV-induced cancers, as well as several cytotoxic and immunomodulatory agents for localized treatment of infections, there are currently no HPV antiviral drugs in our arsenal of therapeutic agents. This review examines the status of past and ongoing research into the development of HPV antivirals, focused primarily upon approaches targeting the replication of the viral genome. The only HPV enzyme, E1, is a DNA helicase that interfaces with the cellular DNA replication machinery to replicate the HPV genome. To date, searches for small molecule inhibitors of E1 for use as antivirals have met with limited success. The lack of other viral enzymes has meant that the search for antivirals has shifted to a large degree to the modulation of protein-protein interactions. There has been some success in identifying small molecule inhibitors targeting interactions between HPV proteins but with activity against a small subset of viral types only. As noted in this review, it is thought that targeting E1 interactions with cellular replication proteins may provide inhibitors with broader activity against multiple HPV types. Herein, we outline the steps in HPV DNA replication and discuss those that appear to provide the most advantageous targets for the development of anti-HPV therapeutics.

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) status in patients with base of tongue cancers., Design: Case-control study using existing patient data., Setting: Roswell Park Cancer Institute., Patients: Thirty patients newly diagnosed with base of tongue squamous cell carcinoma between 1999 and 2005 for whom both tumor samples and periodontal records were available. Patients younger than 21 years, edentulous, immunocompromised, and those with a history of cancer were excluded. Periodontitis history was assessed on the basis of alveolar bone loss (in millimeters) from panoramic radiographs by one examiner who was blinded to cancer status., Main Outcome Measure: HPV-16 and HPV-18 DNA were identified on paraffin-embedded tumor samples by polymerase chain reaction. Multiple logistic regression was used to estimate odds ratios and 95% confidence intervals., Results: The prevalence of tumors positive for HPV-16 DNA was 21 of 30 (70%). None of the samples were positive for HPV-18 DNA. Compared with participants with HPV-negative tumors, patients with HPV-positive tumors had significantly higher mean alveolar bone loss (3.90 mm vs 2.85 mm, P = .01). After adjustment for age at diagnosis, sex, race/ethnicity, alcohol use, smoking status, and number of missing teeth, every millimeter of alveolar bone loss was associated with an approximately 4-fold (odds ratio, 3.96; 95% confidence interval, 1.18-13.36) increased risk of HPV-positive tumor status. Number of missing teeth was not associated with tumor HPV status (odds ratio, 0.95; 95% confidence interval, 0.74-1.21)., Conclusions: Chronic periodontitis may be a significant factor in the natural history of HPV infection in patients with base of tongue cancers. Additional confirmation in larger studies is required.

Proteasome activator PA200 enhances proteasome-mediated cleavage after acidic residues in vitro; however, its role within cells is not known. Here, we show that, in response to ionizing radiation, PA200 forms hybrid proteasomes with 19S caps and 20S core proteasomes that accumulate on chromatin, leading to an increase in proteolytic activity. Unlike many other proteins that respond to DNA damage, the response of PA200 appears to be independent of Ataxia Telangiectasia Mutated and p53, but dependent on DNA-dependent protein kinase activity. Nonetheless, PA200 is critical because PA200-knockdown cells show genomic instability and reduced survival after exposure to ionizing radiation. This phenotype is reproduced by specific inhibition of postglutamyl activity of proteasomes, but combined treatment with PA200 siRNA and postglutamyl inhibitor does not show additive effects on survival. Together, these data suggest a unique role for PA200 in genomic stability that is likely mediated through its ability to enhance postglutamyl cleavage by proteasomes.

Mismatch Repair (MMR) is closely linked to DNA replication; however, other than the role of the replicative sliding clamp (PCNA) in various MMR functions, the linkage between DNA replication and MMR has been difficult to investigate. Here we use an in vitro DNA replication system based on simian virus 40, to investigate MMR recruitment to replicating DNA. Both DNA replication and MMR proteins are recruited to replicating DNA in an origin-dependent fashion. Primer synthesis is required for recruitment of both PCNA and MMR proteins, but not for recruitment of the single-stranded DNA-binding protein (RPA). Blocking PCNA recruitment to replicating DNA with a p21-based polypeptide blocks PCNA and MMR, but not RPA recruitment. Once PCNA and subsequent proteins required for replication are loaded onto DNA, addition of p21 leaves PCNA on the replicating DNA, but actively displaces MMR proteins. These findings indicate that the MMR machinery is recruited to replicating DNA through its interaction with PCNA, and suggests that this occurs via binding of the MMR proteins to the multi-protein interaction sites on PCNA. These studies demonstrate the utility of this system for further investigation of the role of DNA replication in MMR.

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks., (2006 Wiley-Liss, Inc.)

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.

In addition to viral proteins E1 and E2, bovine papillomavirus type 1 (BPV1) depends heavily on host replication machinery for genome duplication. It was previously shown that E1 binds to and recruits cellular replication proteins to the BPV1 origin of replication, including DNA polymerase alpha-primase, replication protein A (RPA), and more recently, human topoisomerase I (Topo I). Here, we show that Topo I specifically stimulates the origin binding of E1 severalfold but has no effect on nonorigin DNA binding. This is highly specific, as binding to nonorigin DNA is not stimulated, and other cellular proteins that bind E1, such as RPA and polymerase alpha-primase, show no such effect. The stimulation of E1's origin binding by Topo I is not synergistic with the stimulation by E2. Although the enhanced origin binding of E1 by Topo I requires ATP and Mg2+ for optimal efficiency, ATP hydrolysis is not required. Using an enzyme-linked immunosorbent assay, we showed that the interaction between E1 and Topo I is decreased in the presence of DNA. Our results suggest that Topo I participates in the initiation of papillomavirus DNA replication by enhancing E1 binding to the BPV1 origin.

The papillomavirus (PV) E2 protein plays a role in recruiting viral and cellular DNA replication factors, such as PV E1 or RPA to PV genomes. Using both purified proteins and through co-precipitation, it was determined that HPV-11 E2 binds human topoisomerase I. E2 can stimulate topoisomerase I DNA relaxation activity 3- to 4-fold. Conversely, topoisomerase I is unable to stimulate E2 DNA binding. These findings suggest that stimulation of topoisomerase I by E2 may help promote efficient relaxation of the torsional stress induced by PV DNA replication.

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Here we show that human RPA70 and RPA32 can be phosphorylated by purified S-phase checkpoint kinases, ATR and Chk1. While ATR phosphorylates the N-terminus of RPA70, Chk1 preferentially phosphorylates RPA's major ssDNA binding domain. Chk1 phosphorylated RPA70 shows reduced ssDNA binding activity, and binding of RPA to ssDNA blocks Chk1 phosphorylation, suggesting that Chk1 and ssDNA compete for RPA's major ssDNA binding domain. ssDNA stimulates RPA32 phosphorylation by ATR in a length dependent manner. Furthermore, 3'-, but not 5'-, recessed single strand/double strand DNA junctions produce an even stronger stimulatory effect on RPA32 phosphorylation by ATR. This stimulation occurs for both RNA and DNA recessed ends. RPA's DNA binding polarity and its interaction to 3'-primer-template junctions contribute to efficient RPA32 phosphorylation. Progression of DNA polymerase is able to block the accessibility of the 3'-recessed ends and prevent the stimulatory effects of primer-template junctions on RPA phosphorylation by ATR. We propose models for the role of RPA phosphorylation by Chk1 in S-phase checkpoint pathways, and the possible regulation of ATR activity by different nucleic acid structures.

The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase alpha-primase, to replicate PV genomes. Here, E1 is shown to bind to human topoisomerase I and stimulate its relaxation activity up to sevenfold. The interaction between E1 and topoisomerase I was mapped to the E1 DNA binding domain and C terminus. These findings imply a mechanism for the recruitment of topoisomerase I to PV DNA replication forks and for stimulating topoisomerase I to allow for efficient relaxation of the torsional stress induced by replication fork progression.

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2). Somewhat higher concentrations resulted in substantial S-phase arrest, and at increasing concentrations, complete cell cycle arrest in G(1) was observed without the appearance of a sub-G(1) cell population. Neither inhibition of cell growth nor cell cycle arrest appeared to be dependent on ataxia and Rad-related kinase expression. DNA damage checkpoint proteins including p53, chk1, and chk2 were differentially activated by hedamycin depending on the concentration and duration of treatment. The level of downstream cell cycle regulators such as cdc25A, E2F1, cyclin E, and p21 were also altered under conditions that induced cell cycle arrest, but atypically, p21 overexpression was observed only in S-phase-arrested cells. Apoptotic indicators were only observed at moderate hedamycin concentrations associated with S-phase arrest, while increasing concentrations, when cells were arrested in G(1), resulted in a reduction of these signals. Taken together, the responses of cells to hedamycin are distinct with regard to its effect on cell cycle but also in the unusual concentration-dependent manner of activation of DNA damage and cell cycle checkpoint proteins as well as the induction of apoptotic-associated events.

With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.

As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses. Low adozelesin concentrations (e.g., 0.5 nM) induced a transient S-phase block and cell cycle arrest in G(2)-M, as well as increased induction of p53 and p21, whereas a high drug concentration (e.g., 2.5 nM) caused apoptosis but no p21 induction. In contrast, both low and high bizelesin concentrations enhanced p53 and p21 induction and triggered G(2)-M cell cycle arrest and eventual senescence without significant apoptotic cell death. However, in cells lacking p21, bizelesin, as well as adozelesin, triggered apoptosis, indicating that p21 was crucial to sustained bizelesin-induced G(2)-M arrest. Thus, despite similar abilities to alkylate DNA, the chemotherapeutic agents adozelesin and bizelesin caused a decrease in HCT116 tumor cell proliferation by different pathways (i.e., adozelesin induced apoptosis, and bizelesin induced senescence).

Adozelesin is an alkylating minor groove DNA binder that is capable of rapidly inhibiting DNA replication in treated cells through a trans-acting mechanism and preferentially arrests cells in S phase. It has been shown previously that in cells treated with adozelesin, replication protein A (RPA) activity is deficient, and the middle subunit of RPA is hyperphosphorylated. The adozelesin-induced RPA hyperphosphorylation can be blocked by the replicative DNA polymerase inhibitor, aphidicolin, suggesting that adozelesin-triggered cellular DNA damage responses require active DNA replication forks. These data imply that cellular DNA damage responses to adozelesin treatment are preferentially induced in S phase. Here, we show that RPA hyperphosphorylation, RPA intranuclear focalization, and gamma-H2AX intranuclear focalization induced by adozelesin treatment are all dependent on DNA replication fork progression, and focalization is only induced in S phase cells. These findings are similar to those seen with the S phase-specific DNA-damaging agent, camptothecin. Conversely, all three DNA damage responses are independent of either S phase or replication fork progression when induced by treatment with the DNA strand scission agent, C-1027. Furthermore, we demonstrate that adozelesin-induced RPA and gamma-H2AX intranuclear foci appear to colocalize within the nuclei of S phase cells.

Treatment of cells with the enediyne C-1027 is highly efficient at inducing single- and double-strand DNA breaks. This agent is highly cytotoxic when used at picomolar levels over a period of days. For this study, C-1027 has been used at higher levels for a much shorter time period to look at early cellular responses to DNA strand breaks. Extracts from cells treated with C-1027 for as little as 2 h are deficient in SV40 DNA replication activity. Treatment with low levels of C-1027 (1-3 nM) does not result in the presence of a replication inhibitor in cell extracts, but they are deficient in replication protein A (RPA) function. Extracts from cells treated with high levels of C-1027 (10 nM) do show the presence of a trans-acting inhibitor of DNA replication. The deficiency in RPA in extracts from cells treated with low levels of C-1027 can be fully complemented by the addition of exogenous RPA, and may be due to a C-1027-induced decrease in the extractability of RPA. This decrease in the extractability of RPA correlates with the appearance of many extraction-resistant intranuclear RPA foci. The trans-acting inhibitor of DNA replication induced by treatment of cells with high levels of C-1027 (10 nM) is DNA-dependent protein kinase (DNA-PK). DNA-PK is activated by the presence of DNA fragments induced by C-1027 treatment, and can be abrogated by removal of the DNA fragments. Although it is activated by DNA damage and phosphorylates RPA, DNA-PK is not required for either RPA focalization or loss of RPA replication activity.

While much has been learned in recent years about the process of chromatin remodeling and its role in activation of transcription, relatively little has been reported on the role of chromatin remodeling in DNA replication. However, it is well established that transcription factors and chromatin structure play an important role in replication origin usage. Recent work has begun to indicate that chromatin remodeling factors are likely to play an important role in the regulation of replication origin usage. The results to date are most consistent with the role for chromatin remodeling factors in DNA replication as being indirect, and very similar to their role in transcription. The current evidence suggests that transcription factors bind to auxiliary sequences adjacent to replication origins and recruit chromatin remodeling factors to create either nucleosome-free regions or regions of specifically spaced nucleosomes. This results in activation of the nearby origin, presumably by making the origin region more accessible to replication factors. Until recently, there has been very little evidence of direct interactions between chromatin remodeling factors and the DNA replication machinery. Recent studies have provided data indicating that direct interactions may exist between chromatin remodeling factors and two cellular replication factors, the Origin Recognition Complex and Proliferating Cell Nuclear Antigen. However, since these replication factors are also involved in other nuclear processes, such as transcriptional silencing and DNA repair, respectively, further study is necessary to establish whether these direct interactions are also important for DNA replication.

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.