RAD51 paralogs synergize with RAD51 to protect reversed forks from cellular nucleases.

Fork reversal is a conserved mechanism to prevent stalled replication forks from collapsing. Formation and protection of reversed forks are two crucial steps in ensuring fork integrity and stability. Five RAD51 paralogs, namely, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which share sequence and structural similarity to the recombinase RAD51, play poorly defined mechanistic roles in these processes. Here, using purified BCDX2 (RAD51BCD-XRCC2) and CX3 (RAD51C-XRCC3) complexes and in vitro reconstituted biochemical systems, we mechanistically dissect their functions in forming and protecting reversed forks. We show that both RAD51 paralog complexes lack fork reversal activities. Whereas CX3 exhibits modest fork protection activity, BCDX2 significantly synergizes with RAD51 to protect DNA against attack by the nucleases MRE11 and EXO1. DNA protection is contingent upon the ability of RAD51 to form a functional nucleoprotein filament on DNA. Collectively, our results provide evidence for a hitherto unknown function of RAD51 paralogs in synergizing with RAD51 nucleoprotein filament to prevent degradation of stressed replication forks.

PARP1 recruits DNA translocases to restrain DNA replication and facilitate DNA repair.

Replication fork reversal which restrains DNA replication progression is an important protective mechanism in response to replication stress. PARP1 is recruited to stalled forks to restrain DNA replication. However, PARP1 has no helicase activity, and the mechanism through which PARP1 participates in DNA replication restraint remains unclear. Here, we found novel protein-protein interactions between PARP1 and DNA translocases, including HLTF, SHPRH, ZRANB3, and SMARCAL1, with HLTF showing the strongest interaction among these DNA translocases. Although HLTF and SHPRH share structural and functional similarity, it remains unclear whether SHPRH contains DNA translocase activity. We further identified the ability of SHPRH to restrain DNA replication upon replication stress, indicating that SHPRH itself could be a DNA translocase or a helper to facilitate DNA translocation. Although hydroxyurea (HU) and MMS induce different types of replication stress, they both induce common DNA replication restraint mechanisms independent of intra-S phase activation. Our results suggest that the PARP1 facilitates DNA translocase recruitment to damaged forks, preventing fork collapse and facilitating DNA repair.

Collision carcinoma of endometrial adenocarcinoma and primary serous fallopian tube carcinoma: a case report.

Collision carcinoma is a rare malignancy that generally occurs in cervical, esophageal, pulmonary, and squamous cell cancers. Few studies have been reported involving endometrial adenocarcinoma and fallopian tube carcinoma. We reported the case of a 58-year-old woman who presented because of irregular vaginal bleeding for more than 1 month. Cervical biopsy suggested moderately differentiated cervical adenocarcinoma, and the patient underwent radical hysterectomy under general anesthesia. However, postoperative pathology and immunohistochemical results indicated a collision tumor comprising endometrial adenocarcinoma (grade I) and primary serous fallopian tube carcinoma. According to the treatment principle of multiple primary tumors, a regimen of paclitaxel combined with carboplatin was administered. The patient also underwent local pelvic radiotherapy to treat lymph node metastasis. One month later, the patient developed brain metastases and died.

[In vitro culture of primary islet cells of suckling rats].

OBJECTIVE: To find a new method for obtaining abundant, well-purified and functionally active rat islet cells cultured in vitro. METHOD: The pancreatic tissues of suckling rats underwent repeated digestion for short durations with collagenase, and the cell growth was observed under inverted microscope 18 h after cell inoculation. The cultured cells were then transferred to a new culture plate and the supernate was harvested regularly to determine the concentration of insulin, amylase and glucose-stimulated insulin release. RESULTS: The fibroblast cells in the primary cultured suckling rat cells were obviously reduced, and the ratio of dithizone-stained cells were 85%-90% with the viability exceeding 90% as assessed by trypan blue staining. The secretion function of the cultured cells remained normal after a 7- to 11-day culture. CONCLUSION: Repeated digestion of the pancreatic tissues for short durations with collagenase and timely cell transfer to new plate may help achieve highly purified viable monolayer islet cells that are well applicable for experimental studies.

[Effect of triglycerides on rat islet cell function in vitro].

OBJECTIVE: To study the effect of triglycerides (TGs) on the function of in vitro cultured rat islet cells. METHODS: SD rat islet cells were isolated for monolayer cell culture and treated with TGs at different concentrations (0, 0.5, 2.5, 5.0, 10.0 mmol/L) for 72 h. Insulin level in the cell culture media was measured after glucose loading test, and the deposition of fat droplets in the islet cells observed by oil red O-staining. RESULTS: No obvious effect was observed after a 72-h incubation of the islet cells with TGs at low glucose level (2.8 mmol/L), while with the stimulation of high-level glucose (27.8 mmol/L), the insulin secretion was significantly inhibited by TGs of higher concentrations (5.0, 10.0 mmol/L). The deposition of fat droplets was found in the islet cells after incubation with TGs, with TGs of higher concentrations. CONCLUSION: TGs induces deposition of fat droplets in islet cells in vitro, and may inhibit the insulin-secreting function of the cells.

[Apoptosis-inducing effect of palmitic acids on rat pancreatic islet cells in primary culture: a preliminary study].

OBJECTIVE: To investigate the in vitro apoptosis-inducing effect of palmitic acid on pancreatic islet cells in primary culture, thereby to understand the role of palmitic acid in the pathogenesis of type 2 diabetes mellitus. METHOD: SD rat pancreatic islet cells were isolated and cultured in monolayer in vitro followed by incubation with stepwise diluted palmitic acid (0, 0.125, 0.25 mmol/L and 0.5 mmol/L respectively), and the insulin concentrations in the culture medium were determined by radio immunological methods. Morphological observation with a fluorescence microscope was conducted after double staining of the cells with PI/Hoechst 33342. RESULTS: The glucose-stimulated insulin secretion was inhibited by palmitic acid at the concentration of 0.25 and 0.5 mmol/L, which also induced obvious cell apoptosis. CONCLUSION: Palmitic acid is capable of inducing islet cell apoptosis in a dose-dependent manner.