The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase eta

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol eta activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only aminimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.ope

Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repairope

CMB-S4

We describe the stage 4 cosmic microwave background ground-based experiment CMB-S4

Analysis of extreme annual rainfall in North-Eastern India using machine learning techniques

The machine learning techniques of Multiple Linear Regression (MLR), Generalized Additive Models (GAMs), and the Random Forest (RF) Method have been used to analyze the extreme annual rainfall in the six states of Assam, Meghalaya, Tripura, Mizoram, Manipur, and Nagaland in North-Eastern (NE) India. Latitude, longitude, altitude, and temperature were the covariates that were used in this study. Ordinary Kriging was used to interpolate the predicted outcomes of each dataset. Statistical metrics like Mean Absolute Errors (MAE), Root Mean Square Error (RMSE), Coefficients of Determination (COD-R2), and Nash–Sutcliffe Efficiency (NSE) were also assessed. When compared to satellite rainfall data, all techniques performed significantly better for ground rainfall data. For prediction, GAM's predicted rainfall values triumph over MLR or RF. RF ranks a close second, while the linearity of MLR prohibits it from making precise predictions for a physical phenomenon like rainfall. The MAE and RMSE of GAM forecasts are significantly lower than those of MLR and RF in most circumstances. Additionally, the COD and NSE of GAM predictions are significantly better than both MLR and RF in most cases, showing that GAM, out of MLR, GAM, and RF, is the best model for predicting rain in our research area. HIGHLIGHTS The map for RF + OK is much more realistic.; The testing (rain gauge data) maps were much better for all the methods than their training counterparts.; Temperature inclusion has an effect on latitude, longitude, and elevation at each point as an attribute.; The use of splines in the linear model allows GAMs to get around linearity constraints.; Comparing the MAE and RMSE of GAM predictions for the various datasets.

Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases

DNA protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide T cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to that of the unmodified control. The DpC induced 20% targeted G -> A and G -> T plus 15% semitargeted mutations, notably at a guanine (G(5)) five bases 3' to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol eta, pol kappa, or pol zeta, indicating that they participate in error-prone bypass of the DpC lesion. However, the semitargeted mutation at G(5) was only reduced upon knockdown of pol zeta, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semitargeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass

Synthesis of Sequence-Specific DNA–Protein Conjugates via a Reductive Amination Strategy

DNA–protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to <i>bis</i>-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their superbulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty in generating site-specific DNA substrates representative of DPC lesions formed <i>in vivo</i>. In the present study, a novel approach involving postsynthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides

Genetic Characterization of Endangered Indian Mithun (<i>Bos frontalis</i>), Indian Bison/Wild Gaur (<i>Bos gaurus</i>) and Tho-tho Cattle (<i>Bos indicus</i>) Populations Using SSR Markers Reveals Their Diversity and Unique Phylogenetic Status

Mithun (Bos frontalis) or gayal and Indian Bison or wild gaur (Bos gaurus) are listed among the rare and endangered bovine species of India. The remote location of mithun in four North Eastern Hill states (Arunachal Pradesh, Nagaland, Manipur, and Mizoram), scattered population size, and non-availability of genetic diversity status are major limitations towards devising a suitable breeding and conservation policy of these species. Since several studies have demonstrated the successful applicability of microsatellite/SSR markers across related genera/families in both crop plants and animal species, 30 FAO recommended cattle microsatellites were utilized for the assessment of the genetic diversity of Indian mithun, bison, and local Tho-tho cattle. Mitochondrial transmembrane protein coding cytochrome B (CYTB) complete sequence data of 71 bovine samples from India were also used to reinforce the study. Population structuring clustered the all bovines into three subgroups as per geographical location and species. Bottleneck analysis indicated a mode shift in the allelic frequency distribution of gaur, indicating minor genetic bottleneck events in the past, while no bottleneck was found in mithun and Tho-tho cattle. To our knowledge, this study represents the first report of molecular genetic characterization showing the population structure and status of genetic diversity in rare Indian bovines, namely, Mithun, Gaur, and Tho-tho cattle

FANCD2-associated Nuclease 1, but Not Exonuclease 1 or Flap Endonuclease 1, Is Able to Unhook DNA Interstrand Cross-links in Vitro

Cisplatin and its derivatives, nitrogen mustards and mitomycin C, are used widely in cancer chemotherapy. Their efficacy is linked primarily to their ability to generate DNA interstrand cross-links (ICLs), which effectively block the progression of transcription and replication machineries. Release of this block, referred to as unhooking, has been postulated to require endonucleases that incise one strand of the duplex on either side of the ICL. Here we investigated how the 5&apos; flap nucleases FANCD2-associated nuclease 1 (FAN1), exonuclease 1 (EXO1), and flap endonuclease 1 (FEN1) process a substrate reminiscent of a replication fork arrested at an ICL. We now show that EXO1 and FEN1 cleaved the substrate at the boundary between the single-stranded 5&apos; flap and the duplex, whereas FAN1 incised it three to four nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing a nitrogen mustard-like ICL two nucleotides in the duplex region because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and, therefore, failed to release the 5&apos; flap. We also show that FAN1 was able to degrade a linear ICL substrate. This ability of FAN1 to traverse ICLs in DNA could help to elucidate its biological function, which is currently unknown.clos