RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models.

BACKGROUND & AIMS: HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. METHODS: Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. RESULTS: HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. CONCLUSIONS: Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. LAY SUMMARY: Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.

Intrinsic ssDNA annealing activity in the C-terminal region of WRN.

Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein.

Werner syndrome (WS) is a genetic premature aging disorder in which patients appear much older than their chronological age. The gene mutated in WS encodes a nuclear protein (WRN) which possesses 3'-5' exonuclease and ATPase-dependent 3'-5' helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as transcription, replication, recombination and repair. Recently we have identified poly(ADP-ribose) polymerase-1 (PARP-1) as a new WRN interacting protein. In this paper, we further mapped the interacting domains. We found that PARP-1 bound to the N-terminus of WRN and to the C-terminus containing the RecQ-conserved (RQC) domain. WRN bound to the N-terminus of PARP-1 containing DNA binding and BRCA1 C-terminal (BRCT) domains. We show that unmodified PARP-1 inhibited both WRN exonuclease and helicase activities, and to our knowledge is the only known WRN protein partner that inactivates both of the WRN's catalytic activities suggesting a biologically significant regulation. Moreover, this dual inhibition seems to be specific for PARP-1, as PARP-2 did not affect WRN helicase activity and only slightly inhibited WRN exonuclease activity. The differential effect of PARP-1 and PARP-2 on WRN catalytic activity was not due to differences in affinity for WRN or the DNA substrate. Finally, we demonstrate that the inhibition of WRN by PARP-1 was influenced by the poly(ADP-ribosyl)ation state of PARP-1. The biological relevance of the specific modulation of WRN catalytic activities by PARP-1 are discussed in the context of pathways in which these proteins may function together, namely in the repair of DNA strand breaks.

Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions.

Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1.

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.

Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation.

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb.