Poly(ADP-ribose)-binding promotes Exo1 damage recruitment and suppresses its nuclease activities

Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5′ flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.     

The MRX complex regulates Exo1 resection activity by altering DNA end structure

Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double‐strand breaks (DSBs). DSB resection requires the Mre11‐Rad50‐Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11‐R10T mutant variant that accelerates DSB resection compared to wild‐type Mre11 by potentiating Exo1‐mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double‐strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.     

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIP^R837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.     

ERCC6L2 mitigates replication stress and promotes centromere stability

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腹腔镜解剖性肝切除治疗肝细胞癌的临床观察

目的:探讨腹腔镜解剖性肝切除治疗肝细胞癌的临床效果及安全性。方法:选择2011年2月~2013年8月在我院进行诊治的肝细胞癌患者90例,将其随机分为治疗组与对照组,每组各45例。治疗组采用腹腔镜解剖性肝切除治疗,对照组采用开腹解剖性肝切除,两组术后都常规化疗3个月,观察和比较两组术中出血量、术后肛门排气时间和术后住院时间,并发症的发生情况及术前后血清谷氨酸转移酶(ALT)与天冬氨酸转移酶(AST)的水平。结果:与对照组相比,治疗组的术中出血量、术后肛门排气时间和术后住院时间均明显降低或缩短(P0.05),术后3个月的膈下积液、切口感染、肺部感染、胆漏的发生率明显降低(P0.05)。两组术前血清ALT与AST值对比差异无统计学意义(P0.05);术后1周,两组的ALT与AST值都明显升上(P0.05);术后3个月,治疗组的ALT与AST值明显低于对照组(P0.05)。所有患者随访到2015年8月,治疗组的中位生存期为(18.33±3.11)个月,而对照组为(12.46±2.19)个月,较治疗组明显缩短(P0.05)。结论:腹腔镜解剖性肝切除治疗肝细胞癌具有更好的微创性,能减少近期并发症的发生,促进肝功能的恢复,且能够延长患者的生存时间。     

Surgery and stem cell niches: An Achilles' heel of metastatic cancer

According to a classic paradigm, surgery only plays a palliative role in patients with metastatic cancer in whom the possibility of a radical resection is non-viable. However, some convincing data proceeding from two randomized trials carried out in patients with metastatic kidney cancer have challenged this assumption. The hypothesis proposes that the resection of the primary tumour in metastatic cancer is the first generation of forthcoming therapies aiming at stem cell niches. It will independently improve the prognosis of metastatic cancer in patients that are fit enough to tolerate major surgery. The phenomenon of late relapses could also be explained on this basis.     

Endocrine testicular function and spermatogenesis persist in calves after partial scrotal resection but not Burdizzo castration

Bull calves for fattening are often castrated during the first weeks of life. Because androgens stimulate growth, there is an interest in males that are infertile but exposed to endogenous testicular steroids. Such a situation occurs in cryptorchids and has been imitated by shortening the scrotum to an extent that the testes are located in a near-inguinal position. In this study, effects of partial scrotal resection (SR) and Burdizzo castration (BZ) on endocrine testicular function, testes histology and on weight at slaughter were studied and compared to orchidectomized (OR) and gonad-intact calves (CO; n = 10 per group; age at castration, 54 ± 3 days; fattening period, 474 ± 11 days). Plasma testosterone concentrations were determined repeatedly, and testes were collected for histopathology at slaughter. We hypothesized that SR inhibits spermatogenesis without loss of testicular steroidogenesis. Group SR animals gained more weight than groups OR and BZ (P < 0.01). Plasma testosterone concentration increased in groups SR and CO (P < 0.01 vs. BZ and OR). Histologically, in all SR animals, testicular and epididymal tissue was identified with a seminiferous epithelium of up to three-cell layers in two animals. Germ cells including elongated spermatids were present in three animals. Shortening of the scrotum thus induced varying degrees of testicular degeneration but 3/10 animals had to be suspected as fertile. In one BZ animal, spermatids were identified whereas in the remaining BZ animals, testes and epididymides consisted of sclerotic fibrous tissue. Partial SR thus induced a cryptorchid-like status but fertility in individual animals must be assumed.     

Use of a vaginal probiotic suppository and antibiotics to influence the composition of the endometrial microbiota

The aim of the present study was to determine the status of the endometrial microbiota in patients with repeated implantation failure (RIF) and to assess treatment strategies for nondominant Lactobacillus (NLD) cases. A total of 392 patients with RIF were enrolled in this prospective cohort study (UMIN-CTR 000038582) and underwent endometrial microbiota analysis. Patients diagnosed with NLD were treated with a combination of oral and vaginal probiotics or oral prebiotics and antibiotics. The outcome was evaluated through re-analysis of the endometrial microbiota following treatment, and the results are presented as cure rates. NLD represented 44.9 % of the total endometrial microbiota in patients with RIF. The most commonly detected bacterium was Gardnerella vaginalis. The cure rates in the oral probiotics + oral prebiotics, antibiotics, oral probiotics + oral prebiotics + antibiotics, vaginal probiotic suppository, and vaginal probiotic suppository + antibiotics groups were 29.5, 33.33, 33.33, 43.6, and 78.6 %, respectively. Significant improvements were noted in the vaginal probiotic suppository + antibiotics group. Moreover, we revealed that approximately half of patients with RIF had NLD. Thus, the combination of a vaginal probiotic suppository and antibiotics may represent an effective treatment for NLD cases.     

Mechanisms of helicase activated DNA end resection in bacteria

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