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DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA. Adoption of DNA as a data storage medium could be accelerated with specialized synthesis processes and codecs. The authors describe TdT-mediated DNA synthesis in which data is stored in transitions between non-identical nucleotides and the use of synchronization markers to provide error tolerance.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3 -/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3 -/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3 -/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3 -/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

To escape replicative senescence, cancer cells have to overcome telomere attrition during DNA replication. Most of cancers rely on telomerase to extend and maintain telomeres, but 4–11% of cancers use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). ALT is prevalent in cancers from the mesenchymal origin and usually associates with poor clinical outcome. Given its critical role in protecting telomeres and genomic integrity in tumor cells, ALT is an Achilles heel of tumors and an attractive target for cancer therapy. Here, we review the recent progress in the mechanistic studies of ALT, and discuss the emerging therapeutic strategies to target ALT-positive cancers.

Abstract In the mid- to late 1970s, recombinant deoxyribonucleic acid methods for cloning and expressing genes in E. coli were under intense development. The important question had become: Can humans design and chemically synthesize novel genes that function in bacteria? This question was answered in 1978 and in 1979 with the successful expression in E. coli of 2 mammalian hormones, first somatostatin and then human insulin. The successful production of human insulin in bacteria provided, for the first time, a practical, scalable source of human insulin and resulted in the approval, in 1982, of human insulin for the treatment of diabetics. In this short review, I give my personal view of how the making, cloning, and expressing of human insulin genes was accomplished by a team of scientists led by Keiichi Itakura, Herbert W. Boyer, and myself. Graphical Abstract Graphical Abstract

Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of the B-family efficiently incorporate nucleotides opposite a number of DNA lesions during translesion DNA synthesis. Primase-polymerase PrimPol and the Pol α-primase complex reinitiate DNA synthesis downstream of the damaged sites using their DNA primase activity. These enzymes can decrease the efficacy of chemotherapy drugs, contribute to the survival of tumor cells and to the progression of malignant diseases. DNA polymerases are promising targets for increasing the effectiveness of chemotherapy, and mutations and polymorphisms in some DNA polymerases can serve as additional prognostic markers in a number of oncological disorders.

Phosphoramidite chemical DNA synthesis technology is utilized for creating de novo ssDNA building blocks and is widely used by commercial vendors. Recent advances in enzymatic DNA synthesis (EDS), including engineered enzymes and reversibly terminated nucleotides, bring EDS technology into competition with traditional chemical methods. In this short study, we evaluate oligos produced using a benchtop EDS instrument alongside chemically produced commercial oligonucleotides to assemble a synthetic gene encoding green fluorescent protein (GFP). While enzymatic synthesis produced lower concentrations of individual oligonucleotides, these were available in half the time of commercially produced oligonucleotides and were sufficient to assemble functional GFP sequences without producing hazardous organic chemical waste.

Background Endometrial cancer (EC) arises from uterine endometrium tissue and is the most prevalent cancer of the female reproductive tract in developed countries. It has been predicted that the global prevalence of EC will increase in part because of its positive association with economic growth and lifestyle. The majority of EC presented with endometrioid histology and mutations in the tumor suppressor gene PTEN, resulting in its loss of function. PTEN negatively regulates the PI3K/Akt/mTOR axis of cell proliferation and thus serves as a tumorigenesis gatekeeper. Through its chromatin functions, PTEN is also implicated in genome maintenance procedures. However, our comprehension of how DNA repair occurs in the absence of PTEN function in EC is inadequate. Methods We utilized The Cancer Genome Atlas (TCGA) data analysis to establish a correlation between PTEN and DNA damage response genes in EC, followed by a series of cellular and biochemical assays to elucidate a molecular mechanism utilizing the AN3CA cell line model for EC. Results The TCGA analyses demonstrated an inverse correlation between the expression of the damage sensor protein of nucleotide excision repair (NER), DDB2, and PTEN in EC. The transcriptional activation of DDB2 is mediated by the recruitment of active RNA polymerase II to the DDB2 promoter in the PTEN-null EC cells, revealing a correlation between increased DDB2 expression and augmented NER activity in the absence of PTEN. Conclusion Our study indicated a causal relationship between NER and EC that may be exploited in disease management.

Background: Genomic integrity is crucial to the cellular life cycle, which involves a tightly regulated process where cells progress through specific phases to ensure that fully replicated, undamaged DNA is inherited by daughter cells. Any dysfunction in this process or unrepaired DNA damage leads to cell cycle arrest and programmed cell death. Cancer cells are known to exploit these mechanisms to continue dividing. Usually, DNA damage arrests replication, allowing the DNA Damage Response (DDR) pathway to activate, which repairs the DNA or bypasses the damage to support cell survival and preserve genome integrity. For DNA damage bypass or translesion synthesis (TLS), a group of low-fidelity polymerases perform error-prone DNA synthesis opposite damaged bases, where REV1 functions as the main scaffolding protein. Previously, we reported non-TLS functions of REV1, including its role in triggering DNA damage-dependent specific DNA metabolic processes. Methods and Results: In this study, we demonstrate that REV1 plays a significant role in cell cycle progression and that its loss causes arrest at the G2/M phase in flow cytometry analysis. This unexpected phenotype includes dysregulation of G2/M regulators, such as Cyclin B1 and tubulins, in REV1-deficient cells compared to controls, as quantified by Western blot. Additionally, phosphorylation of histone H3 at serine 28 was significantly reduced in these REV1-deficient cells. These G2/M arrest features were even more pronounced in REV1-deficient cells treated with the tubulin inhibitor colchicine. Conclusions: Overall, this study reveals a previously unrecognized link between REV1 TLS polymerase inhibition and the G2/M cell cycle arrest.

Background Cisplatin (CDDP) remains a key agent in the treatment of muscle-infiltrating bladder carcinoma (MIBC). However, a proportion of MIBC patients do not respond to chemotherapy, which may be caused by the increased repair of CDDP-induced DNA damage. The purpose of this study was to explore the prognostic value of proteins involved in nucleotide excision repair (NER) and translesion DNA synthesis (TLS) in MIBC patients. Methods This is a retrospective analysis of 86 MIBC patients. The XPA, XPF, XPG, ERCC1, POLI, POLH and REV3L proteins were stained in primary bladder tumors and their levels were analyzed both in the total cohort and in a subgroup with metastatic urothelial carcinoma (mUC) that received gemcitabine and CDDP as a first-line therapy. Both cohorts were divided by percentage of cancer cells stained positive for each protein into subgroups with high and low expression. In the same manner, the combined expression of NER (XPA + ERCC1 + XPF + XPG) and TLS (POLI + POLH + REV3L), as the whole pathways, was analyzed. Results Mortality was 89.5% at the median follow-up of 120.2 months. In the total cohort, patients with tumors stained positive for XPA, XPG and POLI had significantly worse overall survival (OS) compared to those with negative staining [hazard ratio (HR) = 0.60, 0.62 and 0.53, respectively]. Both XPG and POLI were independent prognostic factors in multivariate analyses (MVA). In addition, an increase in NER and TLS pathway expression was significantly associated with worse OS in the total cohort (HR = 0.54 and 0.60, respectively). In the mUC subgroup, high POLI expression was associated with significant deterioration of OS (HR = 0.56) in univariate analyses, and its independent prognostic value was shown in MVA. Conclusions Our study showed significant correlations between the tumor expression of XPG and POLI, as well as NER and TLS as the whole pathways, and inferior OS. Hence, they could constitute prognostic biomarkers and potentially promising therapeutic targets in MIBC. However, a prospective trial is required for further validation, thereby overcoming the limitations of this study.

PrimPol is a human DNA primase and DNA polymerase involved in DNA damage tolerance in both nuclei and mitochondria. PrimPol restarts stalled replication forks by synthesizing DNA primers de novo and also possesses DNA translesion activity (TLS activity). PrimPol efficiently and relatively accurately bypasses several DNA lesions including 8-oxoguanine, thymine glycol and 5-formyluracil. In this work, we showed that PrimPol possesses efficient and accurate TLS activity across 8-oxoadenine, another common DNA lesion caused by oxidative stress. The accuracy of PrimPol on DNA with 8-oxoA was significantly higher compared to DNA containing 8-oxoG. Replacement of Mg2+ ions with Mn2+ stimulated activity of PrimPol on DNA with 8-oxoA and 8-oxoG as well as undamaged A in a sequence-dependent manner by the lesion skipping (or template scrunching) mechanism. Altogether, our data support the idea that PrimPol possesses efficient TLS activity across a wide range of DNA lesions caused by oxidative stress.