The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.
Abundant APOBEC3 (A3) deaminase-mediated mutations can dominate the mutational landscape (‘mutator phenotype’) of some cancers, however, the basis of this sporadic vulnerability is unknown. We show here that elevated expression of the bifunctional DNA glycosylase, NEIL2, sensitizes breast cancer cells to A3B-mediated mutations and double-strand breaks (DSBs) by perturbing canonical base excision repair (BER). NEIL2 usurps the canonical lyase, APE1, at abasic sites in a purified BER system, rendering them poor substrates for polymerase β. However, the nicked NEIL2 product can serve as an entry site for Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. As NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, we suggest that aberrant NEIL2 expression can explain certain instances of A3B-mediated mutations.
Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the double strand break-forming topoisomerase Spo11 and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells lacking the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly
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; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodeling factors mediate centromere dismantlement by Spo11 and Rec8. Remarkably, ectopic expression of either protein in proliferating cells leads to mitotic kinetochore loss in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability, and may jeopardize kinetochores in the increasing number of cancers known to express meiotic proteins.
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