The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm,Helicoverpa armigera (Hubner)

American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha￣?) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶ and 1 × 10⁵ POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.     

The deubiquitinating gene Usp29 is dispensable for fertility in male mice

The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29~(–/–)). Usp29~(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29~(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.     

The Saccharomyces cerevisiae Mlh1-Mlh3 Heterodimer Is an Endonuclease That Preferentially Binds to Holliday Junctions

MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX₂EX₄E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.     

Site-Specific MicroRNA Expression May Lead to Different Subtypes in Ulcerative Colitis

Background and Aim

Ulcerative Colitis (UC) is a type of inflammatory bowel disease, considered as an important disease of gastrointestinal tract having a huge impact on the health of the patient. Prolonged inflammation of colon in UC patients increases the risk of developing colorectal cancer. MiRNA are reported as a connecting link between inflammation and cancer. Differential miRNA expression is reported in Crohn’s disease (CD) patients involving various regions of the gastrointestinal tract. The current study was performed to dissect out the site specific miRNA expression in the colon biopsy samples of UC patients from Northern India.

Methods

Biopsy samples were collected from UC patients and healthy controls from Rectosigmoid Area (RS) and Ascending Colon (AC). MiRNA expression was compared between patients with RS and AC using a microarray platform. Differential expression was further validated by Real Time PCR analysis. Demographic and pathological data of UC -associated CRC patients was collected from the hospital database and analyzed for assessing the site of cancer.

Results

Upon analysis of data generated on a microarray platform and qRT PCR revealed that the expression of six miRNAs hsa-miR-146b-5p, hsa-miR-335-3p, hsa-miR-342-3p, hsa-miR-644b-3p, hsa-miR-491-3p, hsa-miR-4732-3p were downregulated in patients where RS was involved as compared to AC. The expression of hsa-miR-141-3p was upregulated in patients where RS region was involved as compared to AC. Analysis of the registered UC patient’s database from the hospital revealed that the site of CRC was predomimnantly the rectosigmoid region of the colon in most of the cases.

Conclusion

This is the first study to show the differential expression of miRNA involving different sites of colon in UC patients. Taking our data and previous reports into consideration, we propose that differential miRNA expression during UC perhaps contribute in the development of UC-associated CRC at the rectosigmoid area.     

Movement of carabids from grassy strips to crop land in organic agriculture

The colonisation process of carabid beetles from field margins to crop fields under organic agriculture was investigated in 2010 and 2011 in northern Germany in order to find the supporting effects of grassy strips in the agricultural landscape. Displacement and speed of Pterostichus melanarius, Carabus auratus and Poecilus spp. were higher in the crops than in the grassy strips, except for the speed of C. auratus. Among the four species C. auratus, P. melanarius, Pterostichus niger, and Poecilus spp., C. auratus revealed the highest displacement and speed, whereas no significant differences were found between the other three species. P. melanarius showed higher displacement and speed in wheat than in red-clover or pumpkin. Speed and displacement of P. melanarius differed between spring and autumn generations in wheat or red-clover. When they were released at a junction of arable crop and field strips, C. auratus moved indifferently to either arable field or field margins/strips, whereas P. melanarius moved directly to crop centres. At the field margin, Poecilus spp. preferred to move along a grassy strip, while it moved irrespective from the grassy strips in a central site. C. nemoralis and P. niger preferred to move within the grassy strips. Our studies reveal that grassy strips do not affect the dispersal of typical arable field species; however, the species demanding grass vegetation can only overcome the barriers of arable fields either by grassy strips alone or by grassy strips with an adjacent hedge.     

Evolutionarily conserved and testis-specific gene, 4930524B15Rik,is not essential for mouse spermatogenesis and fertility

Molecular Biology Reports - Thousands of genes are involved in spermatogenesis, however, the functional roles of most these genes for male fertility remain to be discovered. This research focused...