Biology subjects

Maizels, N.

Publications and source records attributed to Maizels, N..

6 recordsLinked to original sources

bioRxiv2021

Pathways and signatures of mutagenesis at targeted DNA nicks

Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at targeted nicks. BRCA2 inhibits all categories of mutational events at nicks, including indels, SNVs and HDR. DNA2 and RPA promote 5 resection. Most insertions at nicks consist of a single C incorporated opposite the nick by the translesion polymerase REV1. DNA2 and REV3 inhibit these 1 bp insertions; and DNA2 also inhibits 1 bp deletions. Longer deletions are stimulated by DNA2, REV7 and POLQ. Strikingly, POLQ generates most SNVs at both nicks and double-strand breaks. These results identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ at nicks and highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells.

genetics↗

bioRxiv2020

Rapid, direct detection of bacterial Topoisomerase 1 - DNA adducts by RADAR/ELISA

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

microbiology↗

bioRxiv2019

The "adductome": a limited repertoire of adducted proteins in human cells

Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r=0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins -- the "adductome" -- is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

biochemistry↗

bioRxiv2019

G-quadruplexes sequester free heme in living cells

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, up-regulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme. This identifies a new function for G-quadruplexes and a new mechanism for protection of cells from heme.

genomics↗

bioRxiv2019

DONOR-FREE GENE CORRECTION BY TARGETED INTERHOMOLOG RECOMBINATION

Spontaneous gene correction by interhomolog recombination (IHR) occasionally occurs to ameliorate genetic diseases of blood and skin1-3. Using an engineered endogenous gene as a reporter, we demonstrate that gene correction by IHR is normally infrequent ([≤]0.02%) but is stimulated by DSBs targeted by CRISPR/Cas9 to both homologous chromosomes; reaching frequencies of 0.5%. We further show that depletion of POLQ stimulates IHR frequencies 4-fold, to 2%, and promotes IHR in G2 phase, when recombination between replicated homologs can correct not only compound heterozygous but also autosomal dominant "gain-of-function" mutations, which present a special challenge for gene therapy. The strategies reported here will enable optimization of IHR for gene therapy in a variety of cell types. Advantages include the ability to correct gain-of-function mutations, no need for an exogenous donor, and the potential to limit damage to coding sequence by targeting IHR to introns.

genetics↗

bioRxiv2019

Treatment with 5-aza-dC Induces Apoptotic PARP1-DNA Adducts

The nucleoside analog 5-aza-2-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

biochemistry↗