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Funder: Barncancerfonden; FundRef: http://dx.doi.org/10.13039/501100006313, Funder: Norwegian Research Council; FundRef: http://dx.doi.org/10.13039/501100005416, Funder: Knut och Alice Wallenbergs Stiftelse; FundRef: http://dx.doi.org/10.13039/501100004063, Funder: Cancerfonden; FundRef: http://dx.doi.org/10.13039/501100002794, Funder: Instrumentariumin Tiedes����ti��; FundRef: http://dx.doi.org/10.13039/501100008413, Funder: Science for Life Laboratory; FundRef: http://dx.doi.org/10.13039/501100009252, Funder: Academy of Finland; FundRef: http://dx.doi.org/10.13039/501100002341, Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD+-consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag−/− cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag−/− cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag−/− cells are protected from MMS-induced NAD+ depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag−/− cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD+ dysregulation.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3′end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression., How genome stability is maintained at regions of active transcription is currently not entirely clear. Here, the authors reveal an association between base excision repair factors and transcription elongation to modulate DNA repair.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG; aka MPG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here we show that AAG binds to chromatin and forms complex with active RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes aberrantly methylated bases accumulate towards 3’end, in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide novel insights to maintaining genome stability in actively transcribing chromatin, and reveal roles of aberrantly methylated bases in regulation of gene expression.

The integrity of the genetic information is continuously challenged by numerous genotoxic insults, most frequently in the form of oxidation, alkylation or deamination of the bases that result in DNA damage. These damages compromise the fidelity of the replication, and interfere with the progression and function of the transcription machineries. The DNA damage response (DDR) comprises a series of strategies to deal with DNA damage, including transient transcriptional inhibition, activation of DNA repair pathways and chromatin remodeling. Coordinated control of transcription and DNA repair is required to safeguardi cellular functions and identities. Here, we address the cellular responses to endogenous DNA damage, with a particular focus on the role of DNA glycosylases and the Base Excision Repair (BER) pathway, in conjunction with the DDR factors, in responding to DNA damage during the transcription process. We will also discuss functions of newly identified epigenetic and regulatory marks, such as 5-hydroxymethylcytosine and its oxidative products and 8-oxoguanine, that were previously considered only as DNA damages. In light of these resultsthe classical perception of DNA damage as detrimental for cellular processes are changing. and a picture emerges whereDNA glycosylases act as dynamic regulators of transcription, placing them at the intersection of DNA repair and gene expression modulation.

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic thein vivoenvironment, providingin vitromodels used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previousin vitroexperiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

Many alkylating agents are used as chemotherapeutic drugs and have a long history of clinical application. These agents inflict a wide range of DNA damage resulting in a complex cellular response. After DNA damage, cells trigger a series of signaling cascades promoting cellular survival and cell cycle blockage which enables time for DNA repair to occur. More recently, induction of autophagy has been observed in cancer cells after treatment with different DNA-targeted anticancer drugs, including alkylating agents. Several studies have demonstrated that induction of autophagy after DNA damage delays apoptotic cell death and may therefore lead to chemoresistance, which is the limiting factor for successful chemotherapy. On the other hand, depending on the extent of damage and the cellular context, the induction of autophagy may also contribute to cell death. Given these conflicting results, many studies have been conducted to better define the role of autophagy in cancer cells in response to chemotherapy. In this review, we describe the main alkylating agents used in clinical oncology as well as the cellular response they evoke with emphasis on autophagy.