PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 signaling pathway and to identify genes whose loss-of-function sensitizes cancer cells to PRMT5 inhibition, we performed a CRISPR/Cas9 genetic screen in the presence of a PRMT5 inhibitor. We identified known components of the PRMT5 writer/reader pathway including PRMT5 itself, MEP50/WDR77, PPP4C, SMNDC1 and SRSF3. Interestingly, loss of PRMT1, the major asymmetric arginine methyltransferase, also sensitizes cells to PRMT5 inhibition. We investigated the interplay between PRMT5 and PRMT1, and found that combinatorial inhibitor treatment of small cell lung cancer and pancreatic cancer cell models have a synergistic effect. Furthermore, MTAP -deleted cells, which harbor an attenuated PRMT5–MEP50 signaling pathway, are generally more sensitive to PRMT1 inhibition. Together, these findings demonstrate that there is a degree of redundancy between the PRMT5 and PRMT1 pathways, even though these two enzymes deposit different types of arginine methylation marks. Targeting this redundancy provides a vulnerability for tumors carrying a co-deletion of MTAP and the adjacent CDKN2A tumor suppressor gene.

DNA polymerase ζ (polζ) is critical for bypass of DNA damage and the associated mutagenesis, but also has unique functions in mammals. It is required for embryonic development and for viability of hematopoietic cells, but, paradoxically, skin epithelia appear to survive polζ deletion. We wished to determine whether polζ functions in a tissue-specific manner and how polζ status influences skin tumorigenesis. Mice were produced in which Rev3L (the catalytic subunit of polζ) was deleted in tissues expressing keratin 5. Efficient epidermal deletion of Rev3L was tolerated but led to skin and hair abnormalities, accompanied by evidence of DNA breaks. Unchallenged mice developed tumors in keratin 5-expressing tissues with age, consistent with the chromosomal instability accompanying a polζ defect. Unexpectedly, mice with the Rev3L deletion were much more sensitive to UVB radiation than mice defective in other DNA repair genes. Following irradiation, polζ-defective mice failed to mount skin-regenerative responses and responded to stress by mobilizing melanocytes to the epidermis. However, they did not develop skin tumors after chronic UVB irradiation. To determine the proliferative potential of polζ-deficient skin epithelia, keratinocytes were isolated and examined. These keratinocytes harbored chromosomal gaps and breaks and exhibited a striking proliferation defect. These results can be unified by a model in which slowly dividing cells accumulate replication-associated DNA breaks but otherwise survive Rev3L deletion, but functional polζ is essential for responses requiring rapid proliferation, both in cell culture and in vivo. The results reveal a biological role for mammalian polζ in tolerating DNA damage and enabling proliferative responses in vivo.DNA replication | double-strand breaks | UV radiation | carcinogenesis F ast, efficient genomic duplication requires that DNA be completely intact and in the B form, because replicative DNA polymerases cannot synthesize using a damaged DNA template (1, 2). When such a template is encountered, replication halts, and either a double-strand break (DSB) forms after replication fork collapse or the lesion is bypassed by translesion synthesis (TLS) polymerases or by template switching. After such damage tolerance, the DNA can be repaired. The relative importance of each pathway for maintaining genomic stability and preventing carcinogenesis is unknown.TLS is mediated by specialized DNA polymerases (reviewed in ref.3). DNA polymerase ζ (polζ, catalytic subunit REV3L) stands out as the most important DNA polymerase for bypass of lesions in template DNA. Polζ plays a major role in the bypass of many types of DNA damage, including pyrimidine(6-4)pyrimidone photoproducts induced by UV radiation (4, 5) as well as lesions formed by chemical damaging agents such as cisplatin and benzo[a]pyrene (4). Polζ also can be used for bypass of the frequent endogenously formed abasic sites in DNA (4). In the yeast Saccharomyces cerevisiae, spontaneous and DNA damage-induced mutagenesis is highly ...

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.

Bacteriophage T7 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I. However, in vivo, E. coli DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome. In accord with these roles, T7 DNA polymerase is highly processive while E. coli DNA polymerase I has low processivity. The high processivity of T7 DNA polymerase is achieved through tight binding to its processivity factor, E. coli thioredoxin. We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction. Insertion of this domain into the homologous site in E. coli DNA polymerase I results in a dramatic increase in the processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.High processivity is an important attribute of all replicative DNA polymerases (1). Processivity is defined as the number of nucleotides polymerized by a DNA polymerase during a single association-dissociation cycle with the primer-template. In general, processivity is achieved through the interaction of the DNA polymerase with a class of proteins known as processivity factors.The replication system of phage T7 provides an attractive model for studying the replication of a chromosome, in part due to the economy of the proteins involved. T7 DNA polymerase, the product of the viral gene 5, by itself has low processivity. It dissociates from a primer-template after the incorporation of Ͻ15 nt (2). Upon infection of Escherichia coli, T7 annexes a host protein, thioredoxin, to serve as its processivity factor (3, 4). T7 DNA polymerase and thioredoxin bind in a one-to-one complex with an apparent dissociation constant of 5 nM (5). The binding of thioredoxin to T7 DNA polymerase increases the affinity of the polymerase specifically to a primer-template by 80-fold (6). A consequence of the increased affinity for a primer-template is the ability of T7 DNA polymerase to extend a primer on single-stranded DNA (ssDNA) by thousands of nucleotides without dissociating (2).All known DNA polymerases can be classified into four families on the basis of their amino acid sequence similarities (7). T7 DNA polymerase is a member of the ''Pol I'' family that includes E. coli DNA polymerase I, Thermus aquaticus (Taq) DNA polymerase, mitochondrial DNA polymerase, and the DNA polymerases from phages T5, SP01, and SP02. E. coli DNA polymerase I, the paradigm of this family, is a repair-type DNA polymerase, and as such has low processivity. It dissociates from a primer-template after the incorporation of about 20 nt (8). In contrast to T7 DNA polymerase, it does not associate with any known accessory proteins. The threedimensional structure of the large fragment of E. coli DNA polymerase I (Klenow DNA polymerase) is known (9, 10). The polymerase active site and DNA binding domain are in the carboxyl-terminal half of the molecule, and the 3Ј to 5Ј proofreading exonuclease activity is located in a separ...

High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains from such screens, we propose ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refer to the protein regions associated with a strong sgRNA dropout effect in the screens. Applied to a published CRISPR tiling screen dataset, ProTiler identifies 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlap with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also reveals unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which is validated experimentally. Surprisingly, the CKHS regions are negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR-Cas9 mediated amino acids loss.