FANCM-family branchpoint translocases remove co-transcriptional R-loops

AbstractCo-transcriptional R-loops arise from physiological or aberrant stalling of RNA polymerase, leading to formation of stable DNA:RNA hybrids. Unresolved R-loops can promote genome instability. Here, we show that the Fanconi anemia- and breast cancer-associated FANCM protein can directly unwind DNA-RNA hybrids from co-transcriptional R-loops in vitro. FANCM processively unwinds both short and long R-loops, irrespective of sequence, topology or coating by replication protein A. R-loops can also be unwound in the same assay by the yeast and bacterial orthologs of FANCM, Mph1 and RecG, indicating an evolutionary conserved function. Consistent with this biochemical activity of FANCM, we show that FANCM deficient cells are sensitive to drugs that stabilize R-loop formation. Our work reveals a mechanistic basis for R-loop metabolism that is critical for genome stability.

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DNA Replication Modulates R-loop Levels to Maintain Genome Stability

10.1101/155978 ◽

2017 ◽

Author(s):

Stephan Hamperl ◽

Joshua Saldivar ◽

Michael Bocek ◽

Karlene A. Cimprich

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Genome Stability ◽

Genome Instability ◽

Loop Formation ◽

Origin Firing ◽

Disease States ◽

Replication Fork Progression ◽

Mechanistic Basis ◽

Transcription And Replication

SummaryConflicts between transcription and replication are a potent source of DNA damage. The transcription machinery has the potential to aggravate such conflicts by generating co-transcriptional R-loops as an additional barrier to replication fork progression. Here, we investigate the influence of conflict orientation and R-loop formation on genome stability in human cells using a defined episomal system. This approach reveals that head-on (HO) and co-directional (CD) conflicts induce distinct DNA damage responses. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the CD orientation but promoting their formation in the HO orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings not only uncover an intrinsic function of the replisome in R-loop homeostasis, but also suggest a mechanistic basis for genome instability associated with deregulated DNA replication, which is observed in many disease states, including cancer.

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PHF2 histone demethylase prevents DNA damage and genome instability by controlling cell cycle progression of neural progenitors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903188116 ◽

2019 ◽

Vol 116 (39) ◽

pp. 19464-19473 ◽

Cited By ~ 8

Author(s):

Stella Pappa ◽

Natalia Padilla ◽

Simona Iacobucci ◽

Marta Vicioso ◽

Elena Álvarez de la Campa ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Genome Stability ◽

Cell Cycle Progression ◽

Genome Instability ◽

Neural Progenitors ◽

Cycle Progression ◽

Cycle Arrest ◽

Biochemical Assays

Histone H3 lysine 9 methylation (H3K9me) is essential for cellular homeostasis; however, its contribution to development is not well established. Here, we demonstrate that the H3K9me2 demethylase PHF2 is essential for neural progenitor proliferation in vitro and for early neurogenesis in the chicken spinal cord. Using genome-wide analyses and biochemical assays we show that PHF2 controls the expression of critical cell cycle progression genes, particularly those related to DNA replication, by keeping low levels of H3K9me3 at promoters. Accordingly, PHF2 depletion induces R-loop accumulation that leads to extensive DNA damage and cell cycle arrest. These data reveal a role of PHF2 as a guarantor of genome stability that allows proper expansion of neural progenitors during development.

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Replisome bypass of a protein-based R-loop block by Pif1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020189117 ◽

2020 ◽

Vol 117 (48) ◽

pp. 30354-30361

Author(s):

Grant D. Schauer ◽

Lisanne M. Spenkelink ◽

Jacob S. Lewis ◽

Olga Yurieva ◽

Stefan H. Mueller ◽

...

Keyword(s):

Single Molecule ◽

Genome Stability ◽

Genome Instability ◽

Multiprotein Complex ◽

Genome Maintenance ◽

Visualization Technique ◽

General Displacement ◽

Maintain Genome Stability ◽

Protein Blocks

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstitutedSaccharomyces cerevisiaereplisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

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Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7032 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7032-7042 ◽

Cited By ~ 59

Author(s):

I Antoshechkin ◽

D F Bogenhagen

Keyword(s):

Mitochondrial Dna ◽

Rna Polymerase ◽

Sequence Similarity ◽

Protein A ◽

Mitochondrial Rna ◽

Basal Transcription ◽

Hmg Box ◽

Mitochondrial Rna Polymerase ◽

Dnase I Footprinting

Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.

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Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

Journal of Bacteriology ◽

10.1128/jb.185.6.1808-1816.2003 ◽

2003 ◽

Vol 185 (6) ◽

pp. 1808-1816 ◽

Cited By ~ 5

Author(s):

Victor McAlister ◽

Chao Zou ◽

Robert H. Winslow ◽

Gail E. Christie

Keyword(s):

Rna Polymerase ◽

Serratia Marcescens ◽

Specific Binding ◽

Protein A ◽

Upstream Region ◽

Late Gene Expression ◽

In Vitro Characterization ◽

Template Dna

ABSTRACT NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter P nucA and to the sequence upstream of the P2 late promoter P F is accompanied by DNA bending. NucC protects about 25 nucleotides of the P F upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from P F in vitro.

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High Fidelity of Yellow Fever Virus RNA Polymerase

Journal of Virology ◽

10.1128/jvi.78.2.1032-1038.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 1032-1038 ◽

Cited By ~ 82

Author(s):

Konstantin V. Pugachev ◽

Farshad Guirakhoo ◽

Simeon W. Ocran ◽

Fred Mitchell ◽

Megan Parsons ◽

...

Keyword(s):

Dengue Virus ◽

Rna Polymerase ◽

Yellow Fever ◽

Error Rate ◽

Yellow Fever Virus ◽

Protein A ◽

Fever Virus ◽

Genome Replication ◽

Virus Rna

ABSTRACT Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10−4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10−7 to 2.3 × 10−7. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

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DYNLL1 mis-splicing is associated with replicative genome instability in SF3B1 mutant cells

10.1101/2021.05.26.445839 ◽

2021 ◽

Author(s):

Annie S Tam ◽

Shuhe Tsai ◽

Emily Yun-Chia Chang ◽

Veena Mathew ◽

Alynn Shanks ◽

...

Keyword(s):

Dna Repair ◽

Genome Stability ◽

Genome Instability ◽

Parp Inhibitor ◽

Mutant Cell ◽

Gene Mutations ◽

Suppressor Gene ◽

Tumour Suppressor Gene ◽

Loop Formation ◽

Cancer Hallmarks

Genome instability is a hallmark of cancer that arises through a panoply of mechanisms driven by oncogene and tumour-suppressor gene mutations. Oncogenic mutations in the core splicing factor SF3B1 have been linked to genome instability. Since SF3B1 mutations alter the selection of thousands of 3' splice sites affecting genes across biological pathways, it is not entirely clear how they might drive genome instability. Here we confirm that while R-loop formation and associated replication stress may account for some of the SF3B1-mutant genome instability, a mechanism involving changes in gene expression also contributes. An SF3B1-H662Q mutant cell line mis-splices the 5'UTR of the DNA repair regulator DYNLL1, leading to higher DYNLL1 protein levels, mis-regulation of DNA repair pathway choice and PARP inhibitor sensitivity. Reduction of DYNLL1 protein in these cells restores genome stability. Together these data highlight how SF3B1 mutations can alter cancer hallmarks through subtle changes to the transcriptome.

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Thermodynamically stable and genetically unstable G-quadruplexes are depleted in genomes across species

Nucleic Acids Research ◽

10.1093/nar/gkz463 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6098-6113 ◽

Cited By ~ 25

Author(s):

Emilia Puig Lombardi ◽

Allyson Holmes ◽

Daniela Verga ◽

Marie-Paule Teulade-Fichou ◽

Alain Nicolas ◽

...

Keyword(s):

Negative Selection ◽

Genome Stability ◽

Genome Instability ◽

Regulation Of Gene Expression ◽

Biological Processes ◽

Single Nucleotide ◽

Folding Potential ◽

Species Specific

Abstract G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.

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R-Loop FormationIn Transat an AGGAG Repeat

Journal of Nucleic Acids ◽

10.1155/2013/629218 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Kazuya Toriumi ◽

Takuma Tsukahara ◽

Ryo Hanai

Keyword(s):

Chemical Modification ◽

Rna Polymerase ◽

T7 Rna Polymerase ◽

Loop Formation ◽

Ribonuclease T1 ◽

In Trans ◽

Negative Supercoiling ◽

In Cis

Formation of RNA-DNA hybrid, or R-loop, was studiedin vitroby transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formationin ciswas diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loopin transwith DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling.

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Shared and Group-Specific Features of the Rotavirus RNA Polymerase Reveal Potential Determinants of Gene Reassortment Restriction

Journal of Virology ◽

10.1128/jvi.00409-09 ◽

2009 ◽

Vol 83 (12) ◽

pp. 6135-6148 ◽

Cited By ~ 38

Author(s):

Sarah M. McDonald ◽

Daniel Aguayo ◽

Fernando D. Gonzalez-Nilo ◽

John T. Patton

Keyword(s):

Rna Polymerase ◽

Protein A ◽

Three Dimensional ◽

Structural Data ◽

Homology Model ◽

Recognition Mechanism ◽

Shell Protein ◽

And Function ◽

Gene Reassortment

ABSTRACT Rotaviruses (RVs) are nonenveloped, 11-segmented, double-stranded RNA viruses that are major pathogens associated with acute gastroenteritis. Group A, B, and C RVs have been isolated from humans; however, intergroup gene reassortment does not occur for reasons that remain unclear. This restriction might reflect the failure of the viral RNA-dependent RNA polymerase (RdRp; VP1) to recognize and replicate the RNA of a different group. To address this possibility, we contrasted the sequences, structures, and functions of RdRps belonging to RV groups A, B, and C (A-VP1, B-VP1, and C-VP1, respectively). We found that conserved amino acid residues are located within the hollow center of VP1 near the active site, whereas variable, group-specific residues are mostly surface exposed. By creating a three-dimensional homology model of C-VP1 with the A-VP1 crystallographic data, we provide evidence that these RV RdRps are nearly identical in their tertiary folds and that they have the same RNA template recognition mechanism that differs from that of B-VP1. Consistent with the structural data, recombinant A-VP1 and C-VP1 are capable of replicating one another's RNA templates in vitro. Nonetheless, the activity of both RdRps is strictly dependent upon the presence of cognate RV core shell protein A-VP2 or C-VP2, respectively. Together, the results of this study provide unprecedented insight into the structure and function of RV RdRps and support the notion that VP1 interactions may influence the emergence of reassortant viral strains.

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