DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA-ATP stoichiometrically from its DNA 3′-end to free pol V (UmuD′ 2 C) to form an active mutasome (pol V Mut) with the composition UmuD′ 2 C-RecA-ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA-ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA-ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA-ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.Pol V is a low-fidelity DNA polymerase 1,2 induced as part of the SOS regulon in E. coli in response to DNA damage 3 . The replicative polymerase, pol III, typically stalls when it encounters a DNA template lesion, arresting movement of the replication fork. One pathway that enables restoration of fork movement involves pol V, which replaces pol III on the sliding β-clamp 4-6 and catalyses translesion DNA synthesis (TLS). Pol V copies numerous types of lesions 7 , but in a mutagenic manner 8,9 . After TLS, pol III resumes normal replication.The pol V complex consists of UmuD′ 2 C 10,11 . Both in vitro 1,2,12,13 and in vivo [14][15][16] , pol V activity requires the assembly of an active RecA filament on single-stranded (ss) DNA, termed RecA* 17 . The biological functions of RecA* in strand exchange during homologous recombination and in mediating cleavage of the repressor protein LexA and UmuD during the SOS response are well understood 17 . In contrast, the biochemical role of RecA* in pol-Vdependent mutagenic TLS remains poorly characterized.Proposals for the role of RecA* in TLS have evolved from positioning UmuD′ 2 C on primer/ template (p/t) DNA proximal to a lesion [18][19][20] Formation of activated pol V MutIn earlier studies, either free RecA protein or RecA filament was added to pol V and p/t DNA substrate. Here, we first form RecA* by incubating RecA with biotinylated ssDNA bound to a streptavidin-agarose resin matrix in the presence of ATPγS (adenosine 5′ [γ-thio] triphosphate; see Methods). Pol V (UmuD′ 2 C) is then incubated with RecA* in the absence of p/t DNA, forming an activated pol V species that can be isolated, pol V Mut ( Supplementary Figs 1 and 2). Only once RecA* is removed by centrifugation is p/t DNA added, allowing DNA synthesis in the absence of RecA* (Fig. 1a, b). In these studies, the p/t template is a hairpin with a 3-nucleotide overhang 21 instead of, for example, an oligonucleotide annealed to a ssDNA circle; this prevents formation of activated RecA* on the p/t DNA itself because the hairpin lacks free ssDNA on which RecA* can a...

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, wholetranscriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

SUMMARY Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1E912 mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small molecule tool compound antagonizes ADAR1’s effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSC with active JAK2 signaling.

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′ 2 C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol Vcatalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.

Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve during tumour progression in a manner that conforms to Charles Darwin’s principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the ‘epitranscriptome’) might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli. Deregulation of these epitranscriptomic events contributes to CSC generation and maintenance, which governs cancer progression and drug resistance. In this Review, we discuss the role of malignant RNA processing in CSC generation and maintenance, including mechanisms of RNA methylation, RNA editing and RNA splicing, and the functional consequences of their aberrant regulation in human malignancies. Finally, we highlight the potential of these events as novel CSC biomarkers as well as therapeutic targets.

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30–50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.

DNA–protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an α,β-unsaturated aldehyde, acrolein. DNA–protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA–peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA–peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or dodecylpeptide was covalently attached at the N2 position of guanine via an acrolein adduct, and replication bypass assays carried out with members of the DinB family of polymerases, human polymerase (pol)κ and Escherichia coli (E. coli) pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol κ was able to catalyze both the incorporation and extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all these substrates, suggesting that pol κ would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol κ, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA–peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA–peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N2-guanine linked DNA–peptide cross-links.

Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3 0 UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the 3 0 UTR binding site of tumor suppressor miRNAs.