Project description:Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large scale gene therapy production.
Project description:During the S-phase, conflicts of replication forks with RNA Polymerase II (RNAPII) threaten genomic stability. While the PAF complex can resolve such conflicts during elongation, the particularly deleterious conflicts with stalling RNAPII are resolved by an as of yet unknown mechanism. Here we show that the MYCN oncoprotein forms a ternary complex with RNAPII and the nuclear RNA exosome, a 3’‑5’ exoribonuclease complex. Together with TFIIS, this complex restarts promoter‑proximal RNAPII, allows escape from co-directional transcription-replication conflicts and prevents double‑strand break accumulation. In cells lacking RNA exosome function, MYCN globally terminates transcription. MYCN-mediated termination is triggered by ATM‑dependent recruitment of BRCA1, which then stabilizes nuclear mRNA decapping complexes. Disruption of mRNA decapping activates ATR, indicative of head-on transcription-replication conflicts, and inhibits DNA replication. We propose that MYCN resolves transcription-replication conflicts via this two-step mechanism to sustain the rapid proliferation of neuroendocrine tumor cells.
Project description:During the S-phase, conflicts of replication forks with RNA Polymerase II (RNAPII) threaten genomic stability. While the PAF complex can resolve such conflicts during elongation, the particularly deleterious conflicts with stalling RNAPII are resolved by an as of yet unknown mechanism. Here we show that the MYCN oncoprotein forms a ternary complex with RNAPII and the nuclear RNA exosome, a 3’‑5’ exoribonuclease complex. Together with TFIIS, this complex restarts promoter‑proximal RNAPII, allows escape from co-directional transcription-replication conflicts and prevents double‑strand break accumulation. In cells lacking RNA exosome function, MYCN globally terminates transcription. MYCN-mediated termination is triggered by ATM‑dependent recruitment of BRCA1, which then stabilizes nuclear mRNA decapping complexes. Disruption of mRNA decapping activates ATR, indicative of head-on transcription-replication conflicts, and inhibits DNA replication. We propose that MYCN resolves transcription-replication conflicts via this two-step mechanism to sustain the rapid proliferation of neuroendocrine tumor cells.
Project description:During the S-phase, conflicts of replication forks with RNA Polymerase II (RNAPII) threaten genomic stability. While the PAF complex can resolve such conflicts during elongation, the particularly deleterious conflicts with stalling RNAPII are resolved by an as of yet unknown mechanism. Here we show that the MYCN oncoprotein forms a ternary complex with RNAPII and the nuclear RNA exosome, a 3’‑5’ exoribonuclease complex. Together with TFIIS, this complex restarts promoter‑proximal RNAPII, allows escape from co-directional transcription-replication conflicts and prevents double‑strand break accumulation. In cells lacking RNA exosome function, MYCN globally terminates transcription. MYCN-mediated termination is triggered by ATM‑dependent recruitment of BRCA1, which then stabilizes nuclear mRNA decapping complexes. Disruption of mRNA decapping activates ATR, indicative of head-on transcription-replication conflicts, and inhibits DNA replication. We propose that MYCN resolves transcription-replication conflicts via this two-step mechanism to sustain the rapid proliferation of neuroendocrine tumor cells.
Project description:Transcription is a major obstacle for replication fork progression and transcription-replication collisions constitute a main cause of genome instability. At a genome-wide scale these obstacles can be detected by the accumulation of the replicative Rrm3 helicase required for RF progression through protein obstacles. Here we show that FACT, a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and that also has a role in replication, is needed to resolve transcription-replication conflicts in Saccharomyces cerevisiae. Importantly, ChIP-chip analyses of Rrm3 reveal that replication progression impairment in FACT mutants occur genome-wide, but preferentially at highly transcribed regions. ChIP-chip studies were perfomed with antibodies against Rrm3-FLAG in the yeast S. cerevisiae.
Project description:The malaria parasite Plasmodium falciparum replicates via schizogony: a fundamentally unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. It also has one of the most A/T-biased genomes ever sequenced. Here, we present the first comprehensive study of the specification and activation of DNA replication origins during Plasmodium schizogony. Potential replication origins were found to be abundant, with ORC1-binding sites detected every ~800 bp throughout the genome. They had no motif enrichment, but were biased towards areas of higher G/C content. Origin activation was then measured at single-molecule resolution via DNAscent technology, and was much less dense than ORC1-binding sites, with origins activated preferentially in areas of low transcriptional activity. Consistently, replication forks moved slowest through the most highly transcribed genes, suggesting that conflicts between transcription and origin firing inhibit efficient replication, and that P. falciparum has evolved its S-phase to minimise such conflicts.
Project description:RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, Xenopus RECQL4 has been implicated in initiating DNA replication in oocyte extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (Knock-Out) or expression of a full-length, mutant protein lacking helicase activity (Helicase Dead). Surprisingly, both the RECQL4 Knock-Out and Helicase Dead cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed normal rates in the RECQL4 Knock Out cells, but decreased rates in the RECQL4 inactive cells. Thus, while budding yeast Sld2 is required for DNA replication origin firing, our evidence suggest that human RECQL4 has assumed a role in the regulation of replication fork progression.
Project description:The replication initiation proteins interact dsDNA located at replication origin and ssDNA of DNA unwinding element (DUE), formed as a result of the destabilization of the double-stranded helix of AT-rich origin region. It is a critical step in the DNA replication initiation; however, the structure of nucleoprotein complex involving initiator protein, dsDNA and/or ssDNA is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), structural and bioinformatic analysis, we defined amino acid residues in plasmid Rep proteins, TrfA and RepE, that are essential for interaction with ssDNA. The study of Rep mutant proteins containing single amino acid substitutions affecting DNA interaction reveals the importance of Rep-ssDNA complexes formation for a dsDNA melting at DUE. Furthermore, the crystal structures obtained for complex of RepE protein with DUE ssDNA, and, RepE complexed with both DUE ssDNA and dsDNA containing RepE specific binding site (iteron) revealed that the plasmid initiator can not only bind iterons and ssDNA DUE separately but also can form a tripartite nucleoprotein complex bringing together specific sequences of replication origin. The presented data strongly supports the loop-back model in which a replication initiator molecule interacts with dsDNA and ssDNA.
Project description:Mammalian chromosome replication starts from distinct sites, but the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. We identified a replication initiation determinant (RepID) protein that binds a subset of replication initiation sites. A large fraction of RepID binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from Rep-ID bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication initiation events. These observations are consistent with a model suggesting that RepID facilitates replication initiation at a distinct group of human replication origins.