No abstract

SummaryHistone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

SummaryG-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.

Oxidative DNA damage constitutes a major threat to genetic integrity, and has thus been implicated in the pathogenesis of a wide variety of diseases, including cancer and neurodegeneration. 7,8-dihydro-8oxo-deoxyGuanine (8-oxo-G) is one of the best characterised oxidative DNA lesions, and it can give rise to point mutations due to its miscoding potential that instructs most DNA polymerases (Pols) to preferentially insert Adenine (A) opposite 8-oxo-G instead of the correct Cytosine (C). If uncorrected, A:8-oxo-G mispairs can give rise to C:G→A:T transversion mutations. Cells have evolved a variety of pathways to mitigate the mutational potential of 8-oxo-G that include i) mechanisms to avoid incorporation of oxidized nucleotides into DNA through nucleotide pool sanitisation enzymes (by MTH1, MTH2, MTH3 and NUDT5), ii) base excision repair (BER) of 8-oxo-G in DNA (involving MUTYH, OGG1, Pol λ, and other components of the BER machinery), and iii) faithful bypass of 8-oxo-G lesions during replication (using a switch between replicative Pols and Pol λ). In the following, the fate of 8-oxo-G in mammalian cells is reviewed in detail. The differential origins of 8-oxo-G in DNA and its consequences for genetic stability will be covered. This will be followed by a thorough discussion of the different mechanisms in place to cope with 8-oxo-G with an emphasis on Pol λ-mediated correct bypass of 8-oxo-G during MUTYH-initiated BER as well as replication across 8-oxo-G. Furthermore, the multitude of mechanisms in place to regulate key proteins involved in 8-oxo-G repair will be reviewed. Novel functions of 8-oxo-G as an epigenetic-like regulator and insights into the repair of 8-oxo-G within the cellular context will be touched upon. Finally, a discussion will outline the relevance of 8-oxo-G and the proteins involved in dealing with 8-oxo-G to human diseases with a special emphasis on cancer.

Significance Macromolecules (DNA, proteins, and lipids) in all cells are constantly damaged by reactive oxygen species (ROS). In particular, ROS cause 1,000–7,000 DNA damages per day. Due to its lowest redox potential, the base guanine is mostly affected, resulting in the formation of 8-oxo-7,8-dihydroguanine. This modified base instructs incorporation of adenosine, instead of cytidine, by replicative DNA polymerases, potentially leading to GC→TA transversion mutations. DNA polymerase λ is the most efficient enzyme in performing accurate translesion synthesis over 8-oxo-7,8-dihydroguanine, since it preferentially incorporates the correct cytidine. In this paper we found that the protein called “DNA polymerase δ interacting protein 2” supports DNA polymerase λ in its important task and can protect cells from ROS DNA damage.

The human checkpoint sensor and alternative clamp Rad9-Rad1-Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9-Rad1-Hus1 with MutY DNA glycosylase, DNA polymerase beta and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9-Rad1-Hus1 complex, thus enhancing the quality control of DNA.

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2′-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ’s affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.

Maintenance of genetic stability is crucial for all organisms in order to avoid the onset of deleterious diseases such as cancer. One of the many proveniences of DNA base damage in mammalian cells is oxidative stress, arising from a variety of endogenous and exogenous sources, generating highly mutagenic oxidative DNA lesions. One of the best characterized oxidative DNA lesion is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can give rise to base substitution mutations (also known as point mutations). This mutagenicity is due to the miscoding potential of 8-oxo-G that instructs most DNA polymerases (pols) to preferentially insert an Adenine (A) opposite 8-oxo-G instead of the appropriate Cytosine (C). If left unrepaired, such A:8-oxo-G mispairs can give rise to CG→AT transversion mutations. A:8-oxo-G mispairs are proficiently recognized by the MutY glycosylase homologue (MUTYH). MUTYH can remove the mispaired A from an A:8-oxo-G, giving way to the canonical base-excision repair (BER) that ultimately restores undamaged Guanine (G). The importance of this MUTYH-initiated pathway is illustrated by the fact that biallelic mutations in the MUTYH gene are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). In this review, we will focus on MUTYH, from its discovery to the most recent data regarding its cellular roles and interaction partners. We discuss the involvement of the MUTYH protein in the A:8-oxo-G BER pathway acting together with pol λ, the pol that can faithfully incorporate C opposite 8-oxo-G and thus bypass this lesion in a correct manner. We also outline the current knowledge about the regulation of MUTYH itself and the A:8-oxo-G repair pathway by posttranslational modifications (PTM). Finally, to achieve a clearer overview of the literature, we will briefly touch on the rather confusing MUTYH nomenclature. In short, MUTYH is a unique DNA glycosylase that catalyzes the excision of an undamaged base from DNA.