Prevalent role of homologous recombination in the repair of specific double-strand breaks in Rhizobium etli.

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.

Mechanisms of structural chromosomal rearrangement formation.

Structural chromosomal rearrangements result from different mechanisms of formation, usually related to certain genomic architectural features that may lead to genetic instability. Most of these rearrangements arise from recombination, repair, or replication mechanisms that occur after a double-strand break or the stalling/breakage of a replication fork. Here, we review the mechanisms of formation of structural rearrangements, highlighting their main features and differences. The most important mechanisms of constitutional chromosomal alterations are discussed, including Non-Allelic Homologous Recombination (NAHR), Non-Homologous End-Joining (NHEJ), Fork Stalling and Template Switching (FoSTeS), and Microhomology-Mediated Break-Induced Replication (MMBIR). Their involvement in chromoanagenesis and in the formation of complex chromosomal rearrangements, inverted duplications associated with terminal deletions, and ring chromosomes is also outlined. We reinforce the importance of high-resolution analysis to determine the DNA sequence at, and near, their breakpoints in order to infer the mechanisms of formation of structural rearrangements and to reveal how cells respond to DNA damage and repair broken ends.

A Novel Breakthrough in Leptospira spp. Mutagenesis: Knockout by Combination of CRISPR/Cas9 and Non-homologous End-Joining Systems.

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

Interactions between miRNAs and Double-Strand Breaks DNA Repair Genes, Pursuing a Fine-Tuning of Repair.

The repair of DNA damage is a crucial process for the correct maintenance of genetic information, thus, allowing the proper functioning of cells. Among the different types of lesions occurring in DNA, double-strand breaks (DSBs) are considered the most harmful type of lesion, which can result in significant loss of genetic information, leading to diseases, such as cancer. DSB repair occurs through two main mechanisms, called non-homologous end joining (NHEJ) and homologous recombination repair (HRR). There is evidence showing that miRNAs play an important role in the regulation of genes acting in NHEJ and HRR mechanisms, either through direct complementary binding to mRNA targets, thus, repressing translation, or by targeting other genes involved in the transcription and activity of DSB repair genes. Therefore, alteration of miRNA expression has an impact on the ability of cells to repair DSBs, which, in turn, affects cancer therapy sensitivity. This latter gives account of the importance of miRNAs as regulators of NHEJ and HRR and places them as a promising target to improve cancer therapy. Here, we review recent reports demonstrating an association between miRNAs and genes involved in NHEJ and HRR. We employed the Web of Science search query TS ("gene official symbol/gene aliases*" AND "miRNA/microRNA/miR-") and focused on articles published in the last decade, between 2010 and 2021. We also performed a data analysis to represent miRNA-mRNA validated interactions from TarBase v.8, in order to offer an updated overview about the role of miRNAs as regulators of DSB repair.

A novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systems

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway.

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.

Beyond interstrand crosslinks repair: contribution of FANCD2 and other Fanconi Anemia proteins to the replication of DNA.

Biallelic mutations of FANCD2 and other components of the Fanconi Anemia (FA) pathway cause a disease characterized by bone marrow failure, cancer predisposition and a striking sensitivity to agents that induce crosslinks between the two complementary DNA strands (inter-strand crosslinks-ICL). Such genotoxins were used to characterize the contribution of the FA pathway to the genomic stability of cells, thus unravelling the biological relevance of ICL repair in the context of the disease. Notwithstanding this, whether the defect in ICL repair as the sole trigger for the multiple physiological alterations observed in FA patients is still under investigation. Remarkably, ICL-independent functions of FANCD2 and other components of the FA pathway were recently reported. FANCD2 contributes to the processing of very challenging double strand ends (DSEs: one ended Double Strand Breaks -DSBs- created during DNA replication). Other ICL-independent functions of FANCD2 include prevention of DNA breakage at stalled replication forks and facilitation of chromosome segregation at the end of M phase. The current understanding of replication-associated functions of FANCD2 and its relevance for the survival of genomically stable cells is herein discussed.

Autophagy Roles in the Modulation of DNA Repair Pathways.

Autophagy and DNA repair are biological processes vital for cellular homeostasis maintenance and when dysfunctional, they lead to several human disorders including premature aging, neurodegenerative diseases, and cancer. The interchange between these pathways is complex and it may occur in both directions. Autophagy is activated in response to several DNA lesions types and it can regulate different mechanisms and molecules involved in DNA damage response (DDR), such as cell cycle checkpoints, cell death, and DNA repair. Thus, autophagy may modulate DNA repair pathways, the main focus of this review. In addition to the already well-documented autophagy positive effects on homologous recombination (HR), autophagy has also been implicated with other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Given the relevance of these cellular processes, the clinical applications of drugs targeting this autophagy-DNA repair interface emerge as potential therapeutic strategies for many diseases, especially cancer.

Oxidative stress and mitochondrial dysfunction-linked neurodegenerative disorders.

Reactive species play an important role in physiological functions. Overproduction of reactive species, notably reactive oxygen (ROS) and nitrogen (RNS) species along with the failure of balance by the body's antioxidant enzyme systems results in destruction of cellular structures, lipids, proteins, and genetic materials such as DNA and RNA. Moreover, the effects of reactive species on mitochondria and their metabolic processes eventually cause a rise in ROS/RNS levels, leading to oxidation of mitochondrial proteins, lipids, and DNA. Oxidative stress has been considered to be linked to the etiology of many diseases, including neurodegenerative diseases (NDDs) such as Alzheimer diseases, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Multiple sclerosis, and Parkinson's diseases. In addition, oxidative stress causing protein misfold may turn to other NDDs include Creutzfeldt-Jakob disease, Bovine Spongiform Encephalopathy, Kuru, Gerstmann-Straussler-Scheinker syndrome, and Fatal Familial Insomnia. An overview of the oxidative stress and mitochondrial dysfunction-linked NDDs has been summarized in this review.

Polymorphisms of DNA repair genes are related to the pathogenesis of myelodysplastic syndrome.

Some studies show that alterations in DNA repair genes polymorphisms are associated with the pathogenesis and susceptibility of Myelodysplastic Syndrome (MDS). We genotyped 60 MDS patients for six DNA repair gene polymorphisms: BRCA1 rs4793191, BRCA2 rs9567623, RAD51 rs1801320, XRCC5 rs3835, XRCC6 rs2267437 and LIG4 rs1805388. The G/C heterozygote genotype of rs1801320 polymorphism was associated with a decreased chance of developing MDS (p = 0.05). Additionally, the G/G homozygous genotype was associated with the presence of one cytopenia in whole blood. The genotype C/G and CG + GG of the rs2267437 polymorphism was associated with normal karyotype (p = 0.010) and bone marrow cellularity normocellular + hypercellular (p = 0.023). We found that the A/G heterozygous genotype of the rs3835 polymorphism is associated with decreased chance of developing MDS (p < 0.001). These results support the importance of RAD51, XRCC5 and XRCC6 genes polymorphisms in the maintenance of genomic stability promoting a better understanding of the genesis and etiology of MDS.

Ionizing radiation-induced gene expression changes in TP53 proficient and deficient glioblastoma cell lines.

The genetic heterogeneity presented by different cell lines derived from glioblastoma (GBM) seems to influence their responses to antitumoral agents. Although GBM tumors present several genomic alterations, it has been assumed that TP53, frequently mutated in GBM, may to some extent be responsible for differences in cellular responses to antitumor agents, but this is not clear yet. To directly determine the impact of TP53 on GBM response to ionizing radiation, we compared the transcription profiles of four GBM cell lines (two with wild-type (WT) TP53 and two with mutant (MT) TP53) after 8Gy of gamma-rays. Transcript profiles of cells analyzed 30 min and 6h after irradiation showed that WT TP53 cells presented a higher number of modulated genes than MT TP53 cells. Our findings also indicate that there are several pathways (apoptosis, DNA repair/stress response, cytoskeleton organization and macromolecule metabolic process) in radiation responses of GBM cell lines that were modulated only in WT TP53 cells (30 min and 6h). Interestingly, the majority of differentially expressed genes did not present the TP53 binding site, suggesting secondary effects of TP53 on transcription. We conclude that radiation-induced changes in transcription profiles of irradiated GBM cell lines mainly depend on the functional status of TP53.

The distribution of recombination repair genes is linked to information content in bacteria.

The concept of a 'proteomic constraint' proposes that the information content of the proteome exerts a selective pressure to reduce mutation rates, implying that larger proteomes produce a greater selective pressure to evolve or maintain DNA repair, resulting in a decrease in mutational load. Here, the distribution of 21 recombination repair genes was characterized across 900 bacterial genomes. Consistent with prediction, the presence of 17 genes correlated with proteome size. Intracellular bacteria were marked by a pervasive absence of recombination repair genes, consistent with their small proteome sizes, but also consistent with alternative explanations that reduced effective population size or lack of recombination may decrease selection pressure. However, when only non-intracellular bacteria were examined, the relationship between proteome size and gene presence was maintained. In addition, the more widely distributed (i.e. conserved) a gene, the smaller the average size of the proteomes from which it was absent. Together, these observations are consistent with the operation of a proteomic constraint on DNA repair. Lastly, a correlation between gene absence and genome AT content was shown, indicating a link between absence of DNA repair and elevated genome AT content.

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80ºC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAiTM was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1