Biology subjects

Tonzi, P.

Publications and source records attributed to Tonzi, P..

4 recordsLinked to original sources

bioRxiv2020

Fused in sarcoma regulates DNA replication timing and progression

Fused in sarcoma (FUS) encodes a low complexity RNA-binding protein with diverse roles in transcriptional activation and RNA processing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS has also been implicated in DNA double-strand break repair (DSBR) and genome maintenance. However, the underlying mechanisms are unknown. Here we employed quantitative proteomics, transcriptomics, and DNA copy number analysis (Sort-Seq), in conjunction with FUS-/- cells to ascertain roles of FUS in genome protection. FUS-/- cells exhibited alterations in the recruitment and retention of DSBR factors BRCA1 and 53BP1 but were not overtly sensitive to genotoxins. By contrast, FUS-deficient cells had reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of pre-replication complexes, and attenuated expression of S-phase associated genes. FUS interacted with lagging strand DNA synthesis factors and other replisome components, but did not translocate with active replication forks. Using a Sort-Seq workflow, we show that FUS contributes to genome-wide control of DNA replication timing and is essential for the early replication of transcriptionally active DNA. These findings illuminate new roles for FUS in DNA replication initiation and timing that may contribute to genome instability and functional defects in cells harboring disease-associated FUS fusions.

genomics↗

bioRxiv2019

Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity. Author summaryIncreasing evidence suggest that instabilities at common fragile sites (CFSs), breakage-prone genomic loci, may be source of genomic aberration seen in cancer cells. Among the proposed mechanisms that can cause CFSs instabilities is the conflict between transcription and replication, and the mechanisms or factors that resolve the possible conflicts are only beginning to be understood. Here we found that deficiency in the Polycomb group proteins BMI1 or RNF2 leads to the CFS instability, and is associated with transcription-associated replication fork stresses. We further found that in the absence of RNF2, cells depend on the Fanconi Anemia fork-protective proteins for genome maintenance and survival. These results underscore that the Polycomb proteins are important for genome maintenance.

cell biology↗

bioRxiv2019

Y-Family DNA Polymerases Have Three Structural Elements That Promote Accurate dCTP Insertion And Minimize dATP/dGTP/dTTP Misinsertion Opposite a N2-dG Adduct of Benzopyrene

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP {kappa} and DNAP IV, which accurately insert dCTP opposite N2-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP {eta} and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N2-dG. To investigate structural differences between Y-Family-classes, Dpo4 (a canonical {eta}/V-class-member) is modified to make it more {kappa}/IV-like, as evaluated via primer-extension studies with a BP-N2-dG-containing template. Three protein structural elements are identified that promote fidelity. (1) Watson-Crick-like [dCTP:BP-N2-dG] pairing requires the BP-moiety to be in the minor groove. Thus, as expected, dCTP insertion is facilitated by having large openings in the protein surface that can accommodate BP-bulk in the minor groove. The BP-moiety is also in the minor groove during dATP and dTTP misinsertion, though evidence suggests that each of these three minor groove BP-conformations differ. (2) Plugging an opening on the major groove side of the protein suppresses dGTP misinsertion, implying BP-N2-dG bulk is in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. (3) Y-Family DNAPs have non-covalent bridges (NCBs) holding their little finger-domain in contact with their catalytic core-domain; dATP/dGTP/dTTP misinsertions are suppressed by the quantity and quality of NCBs, including one near and another distal to the active site on the minor groove side. In conclusion, three protein structural elements enhance dCTP and/or suppress dATP/dGTP/dTTP insertion; four different BP-adduct conformations are responsible for the four different dNTP insertional pathways opposite BP-N2-dG; generalizations about Y-Family structure are also considered.

molecular biology↗

bioRxiv2018

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

molecular biology↗