Protein ubiquitination and deubiquitination are central to the control of a large
number of cellular pathways and signaling networks in eukaryotes. Although the essential
roles of ubiquitination have been established in the eukaryotic DNA damage response, the
deubiquitination process remains poorly defined. Chemical probes that perturb the activity
of deubiquitinases (DUBs) are needed to characterize the cellular function of
deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1
deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase,
deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the
cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into
the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia
pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer
and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA
damage response and a target for overcoming resistance to the platinum-based anticancer
drugs.
The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N-and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle.
Representing a basal branch of arachnids, scorpions are known as ‘living
fossils’ that maintain an ancient anatomy and are adapted to have survived
extreme climate changes. Here we report the genome sequence of Mesobuthus
martensii, containing 32,016 protein-coding genes, the most among sequenced
arthropods. Although M. martensii appears to evolve conservatively, it has a
greater gene family turnover than the insects that have undergone diverse morphological
and physiological changes, suggesting the decoupling of the molecular and morphological
evolution in scorpions. Underlying the long-term adaptation of scorpions is the
expansion of the gene families enriched in basic metabolic pathways, signalling
pathways, neurotoxins and cytochrome P450, and the different dynamics of expansion
between the shared and the scorpion lineage-specific gene families. Genomic and
transcriptomic analyses further illustrate the important genetic features associated
with prey, nocturnal behaviour, feeding and detoxification. The M. martensii
genome reveals a unique adaptation model of arthropods, offering new insights into the
genetic bases of the living fossils.
DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N 2 -dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N 2 -(1-carboxyethyl)-2-deoxyguanosine (N 2 -CEdG) as the major stable DNA adduct. Here, we found that N 2 -CEdG could be detected at a frequency of one lesion per 10 7 nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N 2 -CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N 2 -CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N 2 -CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase ), opposite the N 2 -CEdG is both accurate and efficient. Taken together, our data support that N 2 -CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.glycolysis ͉ mutagenesis ͉ polymerase ͉ translesion synthesis
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