Thomas Melendy

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.

Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

ABSTRACT 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.

The E1 proteins

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.

Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability

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.

Chronic periodontitis-human papillomavirus synergy in base of tongue cancers.

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.

The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

ABSTRACT 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.

Mismatch Repair proteins are recruited to replicating DNA through interaction with Proliferating Cell Nuclear Antigen (PCNA)

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.

Papillomavirus E1 Protein Binds to and Stimulates Human Topoisomerase I

ABSTRACT The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase α-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.

Human papillomavirus DNA replication

Publisher Summary Human papillomaviruses (HPV) are a family of double-stranded circular DNA viruses with a genomic size of around eight kilobases. HPV are highly species–specific and infect only stratified squamous epithelial cells or mucosal membranes. Similar to many other aspects of HPV molecular biology, the DNA replication of bovine papillomavirus type 1 (BPV) has been the most studied and has become the paradigm for HPV–DNA replication. DNA replication studies with various HPVs have shown that although the BPV paradigm is often accurate, differences do exist between the viral DNA replication of BPV and some HPVs. Viruses infect at the basal layer of epidermal tissue and the virus undergoes three stages of replication. Within the basal and first suprabasal epidermal layers, the viral DNA is propagated until there are 50–100 copies per cell. During the second stage, the maintenance stage, viral DNA replication is replicated in synchrony with the host cell cycle, only occurring during S phase of the host cell cycle. During both of these stages the HPV DNA is maintained as episomal plasmids in the nuclei of infected cells. The third and final stage is the vegetative stage of viral replication. The vegetative stage only occurs in terminally differentiated tissues, and includes both an increase in the number of viral genome copies, as well as the expression of late genes and assembly of new virus.. The chapter also discusses E1 protein binding sequence (E1BS), E2 protein family, HPV–DNA replication in terminally differentiated cells, and so on.

Adozelesin Triggers DNA Damage Response Pathways and Arrests SV40 DNA Replication through Replication Protein A Inactivation

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect onin vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.