BRCA1 Interactors - Rad50 and BRIP1: As Prognostic and Predictive Molecules for the Severity of Triple-Negative Breast Cancer

BRIP1 is one of the major interacting partner of BRCA1 which plays an important role in repair by homologous recombination (HR). This gene is mutated in around 4% cases of breast cancer, however, its mechanism of action is unclear. In this study, we presented the fundamental role of BRCA1 interactors BRIP1 and RAD50 in the development of differential severity in Triple-Negative Breast Cancer(TNBC) among various affected individuals. We showed that in some TNBC lines like MDA-MB-231 the functioning of both BRCA1/TP53 is compromised. Furthermore, the sensing of DNA damage is affected, depicted through the low expression of damage sensing molecule Rad50 and reduced formation of H2AX foci. Due to less damage sensing capability and low availability of BRCA1 at the damage sites, the repair by HR becomes inefficient leading to more damage. Accumulation of damage sends a signal for over activation of NHEJ repair pathways. Over expressed NHEJ molecules with compromised HR and checkpoint conditions lead to higher proliferation and error-prone repair, which increases the mutation rate and corresponding tumor severity. The severity phenotypes were more in cells having compromised BRCA1-BRIP1 functioning. The in silico analysis of the TCGA-UCSC xena datasets with genes expression in deceased population shows a significant correlation of BRCA1 expression with OS in TNBCs (0.0272). The association of BRCA1 with OS becomes stronger with the addition of BRIP1expression (0.000876**). Since the overall survival(OS) is directly proportional to the extent of severity, the data analysis hints at the role of BRIP1 in controlling the severity of TNBC.

Enhancement of Indigenous Fungal Cellulase Production by Gamma Rays

The increasing demand for cellulases causes the need for a high cellulase-producing microbe. Mutagenesis is an efficient way to produce a high-titer cellulase-producing strain. Mutagenesis using gamma rays irradiation has the advantage that it can cause a double strand break of DNA. Repair of double-strand break tends to has an error-prone repair that leads to the alteration of DNA sequence. The aim of this study was to screen high cellulase-producing indigenous fungal mutants produced by mutagenesis. Trichoderma sp. PK1J2 was subjected to gamma irradiation at 300 Gy. The mutants produced were screened using a plate medium containing cellulose as a sole carbon source. After staining with congo red, colonies with wider clear zones were grown in a liquid medium for four days, and the cellulase activities were analyzed. Mutant M8 produces endoglucanase, FPase, and β-glucosidase at 0.46 U/ml, 0.18 U/ml, and 1.10 U/ml, respectively, which were 90%, 50%, and 30% higher than those of the parental strain.

Genomic instability is an early event driving chromatin reorganization and escape from oncogene-induced senescence

SUMMARYOncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not timely removed via immune surveillance, senescent cells will also present a detrimental side. Although this has mostly been attributed to the senescence-associated-secretory-phenotype (SASP) of these cells, we recently proposed that “escape” from the senescent state represents another unfavorable outcome. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion, which harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and for driving cell cycle re-entry and malignant transformation. In summary, we now provide strong evidence in support of genomic instability underlying “escape” from oncogene-induced senescence.HIGHLIGHTSOncogene driven error-prone repair produces early genetic lesions allowing escape from senescenceCells escaping oncogene-induced senescence display mutational signatures observed in cancer patientsA single recurrent inversion harboring a circadian TF gene suffices for bypassing oncogene-induced senescenceChromatin loop and compartment remodeling support the “escape” transcriptional program

DNA damage bypass pathways and their effect on mutagenesis in yeast

ABSTRACT What is the origin of mutations? In contrast to the naïve notion that mutations are unfortunate accidents, genetic research in microorganisms has demonstrated that most mutations are created by genetically encoded error-prone repair mechanisms. However, error-free repair pathways also exist, and it is still unclear how cells decide when to use one repair method or the other. Here, we summarize what is known about the DNA damage tolerance mechanisms (also known as post-replication repair) for perhaps the best-studied organism, the yeast Saccharomyces cerevisiae. We describe the latest research, which has established the existence of at least two error-free and two error-prone inter-related mechanisms of damage tolerance that compete for the handling of spontaneous DNA damage. We explore what is known about the induction of mutations by DNA damage. We point to potential paradoxes and to open questions that still remain unanswered.

DNMT3b Dysfunction Promotes DNA cleavages at Centromeric R-loops to Increase Centromere Instability

ABSTRACTThis study investigates how DNA methyltransferase 3b (DNMT3b) dysfunction causes genome instability. We showed that in DNMT3b deficient cells, R-loops contribute to prominent γH2AX signal, which was mapped to repetitive satellite sequences including centromere regions. By ChIP and DRIP analyses, our data revealed that centromeric R-loops in DNMT3b deficient cells are removed by XPG/XPF, thus generating DNA breaks in centromeres to increase mitotic aberration. In immunodeficiency-centromeric instability-facial anomalies (ICF) patient cells carrying the loss-of-function mutation at DNMT3b, knockdown of XPG/XPF in ICF cells also reduces DNA breaks in centromere while bringing up centromeric R-loop to the level similar to that in wild-type cells. These results suggest that DNMT3b has a critical function in preventing XPG/XPF-mediated cleavages at centromeric R-loop sites. Finally, we showed the involvement of non-homologous end-joining repair at centromeric sites in ICF cells. Thus, DNA cleavages at centromeric R-loops with error-prone repair undermine centromere stability in ICF cells.

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis

ABSTRACT We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in Bacillus subtilis YB955 (hisC952 metB5 leuC427). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed B. subtilis YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, mfd and greA were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of mfd or greA abrogated the production of stress-associated amino acid revertants in the GO and nfo exoA nth strains, respectively. These results suggest that in nutritionally stressed B. subtilis cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase ΔytkD ΔmutM ΔmutY (referred to here as ΔGO) and Δnfo ΔexoA Δnth (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of mfd or greA increased the production of His+, Met+, or Leu+ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of B. subtilis. IMPORTANCE In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed B. subtilis strain YB955 (hisC952 metB5 leuC427) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing B. subtilis to escape from growth-limiting conditions.

The main problem of studying biological evolution

The evolutionary aspect of the emergence problem is the main problem of studying the laws of biological evolution. A possible mechanism of how an ontogenetically modified phenotype (phenotypic modification, “morphosis”) is fixed in the genotype – the problem of “genetic assimilation” is considered. In particular, it is assumed that adaptive mutagenesis is involved in this, generating random multiple mutations that are “not Lamarckian”, but Darwinian, because they occur in random places in the genome. Stress-induced mutations that arise as a result of error-prone repair processes, while not targeting specific genes, are not randomly scattered around the genome. On the contrary, these mutations are concentrated around double-stranded DNA breaks caused by various stressors. It is assumed that the breaks occur with greater probability in actively transcribed DNA regions, reflecting the current activity of the organism and being the most open DNA regions. All this creates the conditions for the more likely appearance of useful mutations in the “trained” locus of the genome. Keywords: selectogenesis, nomogenesis, genetic assimilation, stress-mutagenesis.

FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair

ABSTRACT Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

Making targeted gene deletions is essential for studying organisms, but is difficult in many prokaryotes due to the inefficiency of homologous recombination based methods. Here, I describe an easily modifiable, single-plasmid system that can be used to make rapid, sequence targeted, markerless knockouts in both a Gram-negative and a Gram-positive organism. The system is comprised of targeted DNA cleavage by Cas9 and error-prone repair by Non-Homologous End Joining (NHEJ) proteins. I confirm previous results showing that Cas9 and NHEJ can make knockouts when NHEJ is expressed before Cas9. Then, I show that Cas9 and NHEJ can be used to make knockouts when expressed simultaneously. I term the new method Potentially Organism-Agnostic Knockout (POAK) system and characterize its function in Escherichia coli and Weissella confusa. First, I develop a novel transformation protocol for W. confusa. Next, I show that, as in E. coli, POAK can create knockouts in W. confusa. Characterization of knockout efficiency across galK in both E. coli and W. confusa showed that while all gRNAs are effective in E. coli, only some gRNAs are effective in W. confusa, and cut site position within a gene does not determine knockout efficiency for either organism. I examine the sequences of knockouts in both organisms and show that POAK produces similar edits in both E. coli and W. confusa. Finally, as an example of the importance of being able to make knockouts quickly, I target W. confusa sugar metabolism genes to show that two sugar importers are not necessary for metabolism of their respective sugars. Having demonstrated that simultaneous expression of Cas9 and NHEJ is sufficient for making knockouts in two minimally related bacteria, POAK represents a hopeful avenue for making knockouts in other under-utilized bacteria.