Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine–arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.DNA double-stranded breaks (DSBs)³ can be produced in physiological and genotoxic processes. Improper repair or failure to repair DSBs can lead to gene deletions, duplications, translocations, and missegregation of large chromosome fragments, which may result in gene dosage imbalance, cancer development, or cell death (1–3). Historically, two distinct pathways have been described which ensure that DSBs are repaired: nonhomologous end joining (NHEJ) and homologous recombination (HR). During HR, the damaged chromosome interacts via synapsis with an undamaged DNA molecule with which it shares extensive sequence homology, usually its sister chromatid (4, 5). HR is most active in the late S and G₂ phases of the cell cycle. In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6, 7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8, 9).Two types of end-joining reactions can be defined operationally. The first one, which may be called conservative NHEJ (C-NHEJ), is characterized by the precise joining of short, overhanging, complementary ends. Proteins including Ku70/Ku80 and XRCC4 (10–12) are associated with this highly efficient pathway, whereby most ends are rejoined successfully without any alteration of the DNA sequence (8). The alternative pathway for NHEJ is the highly mutagenic and deletional NHEJ (D-NHEJ), which results in short deletions after use of imperfect microhomology of about 1–10 bp at the repair junctions. D-NHEJ activity has been demonstrated in the budding yeast Saccharomyces cerevisiae. In addition, D-NHEJ is independent of Rad52, Rad1, or Ku80 but depends on Mre11 in yeast (13, 14). However, the genetic determinants of this subpathway have not been well established in mammalian cells.Mre11 is the core subunit of the Mre11·Rad50·Nbs1 complex (called the MRN complex), which is conserved throughout all kingdoms of life. The MRN complex is a central player in most aspects of the cellular response to DSBs, including HR, NHEJ, telomere maintenance, and DNA damage checkpoints (15–17). Loss of Mre11 results in increased radiosensitivity and chromosomal instability (17). Patients with germ line mutations of Mre11 have clinical presentations similar to those of ataxia telangiectasia patients (ataxia telangiectasia-like disorder) (18).After DNA damage, the MRN complex is recruited to the sites of damage via zinc hooks at the ends of the long, flexible arms of Rad50 (19, 20). Mre11 contains both single-stranded DNA endonuclease and 3′-5′ exonuclease activities in vitro, but in vivo Mre11 is also implicated in 5′-3′ DSB resection. The MRN complex also interacts with BRCA1 and CtIP, which may be essential for DSB end resection to generate 3′ overhanging single-stranded DNA during initiation of HR (21, 22).Mre11 has an N-terminal nuclease domain, which contains five phosphoesterase motifs, and a C-terminal glycine–arginine-rich domain (GAR). Arthur et al. (23) showed that an H85L mutation completely abrogated exonuclease activity, whereas binding to Rad50 and Nbs1 was retained. Complementation of ataxia telangiectasia-like disorder cells with this mutant, called Mre11-3, restored the localization of the MRN complex to DSBs in IR-induced foci (23, 24). Methylation of the GAR region has also been shown to be important for the DNA binding and exonuclease activity of Mre11 in vitro (25, 26). Both the crystal structure of yeast Mre11 and data from conditional knock-out mice (Mre11^H129N/Δ) reveal that the nuclease activity of Mre11 is required for HR repair of DSBs (22, 27). However, the role of Mre11 in NHEJ is not well defined (27, 28). Most recently, Mre11 was reported to support NHEJ in mammalian cell (29–31). However, whether Mre11 regulates both NHEJ subpathways or only D-NHEJ is controversial, and the mechanisms by which Mre11 is involved in NHEJ remain to be established.To address these questions, we have established a system that can analyze the accuracy and efficiency of rejoining of two adjacent DSB ends at chromosomal level in human embryonic kidney 293 (HEK293) cells. We show here that Mre11 siRNA knockdown in these cells results in significant reduction of the overall NHEJ efficiency. Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-NHEJ by ∼10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of C-NHEJ. Mutation of Mre11 in either the phosphoesterase domain (Mre11-3) or the GAR region (Mre11-R/A) to produce abnormal exonuclease activity impaired the D-NHEJ pathway only. The D-NHEJ deficiency is significantly more severe in cells with Mre11-R/A than that in cells with Mre11-3. Therefore, our data suggest that Mre11 is required specifically for D-NHEJ repair of DNA DSBs and that its exonuclease activity is at least one of the important mechanisms for this DNA end joining subpathway.     

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant <Emphasis Type=Italic>Lactococcus lactis</Emphasis> for oral vaccination and its protective immune response in mice

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C₈₃₅₄₉ challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice. An erratum to this article can be found at     

粤北普通野生稻籼粳分化的SSR分析

本文利用28对籼粳特异性SSR引物对174个粤北普通野生稻个体进行分析.28对引物均有多态性,平均每对引物扩增出的等位变异数和基因型数分别为10.6和28.粤北普通野生稻群体有较高的遗传多样性(0.768 1),除了能扩增出典型的籼、粳特异基因,还能扩增出野生稻特有的基因.遗传一致度聚类分析表明粤北普通野生稻存在初步籼粳分化;2份(1.15%)材料偏籼,172份(98.85%)材料偏粳,群体以偏粳为主.基因组水平上整个粤北群体没有发现原始类型.     

A quantitative assay measuring the function of lipase maturation factor 1

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression.     

Development of an enrichment culture growing at low temperature used for ensiling rice straw

To speed up the conversion of rice straw into feeds in a low-temperature region, a start culture used for ensiling rice straw at low temperature was selected by continuous enrichment cultivation. During the selection, the microbial source for enrichment was rice straw and soil from two places in Northeast China. Lab-scale rice straw fermentation at 10 degrees C verified, compared with the commercial inocculant, that the selected start culture lowered the pH of the fermented rice straw more rapidly and produced more lactic acid. The results from denatured gradient gel eletrophoresis showed that the selected start culture could colonize into the rice straw fermentation system. To analyze the composition of the culture, a 16S clone library was constructed. Sequencing results showed that the culture mainly consisted of two bacterial species. One (A) belonged to Lactobacillus and another (B) belonged to Leuconostoc. To make clear the roles of composition microbes in the fermented system, quantitative PCR was used. For species A, the DNA mass increased continuously until sixteen days of the fermentation, which occupied 65%. For species B, the DNA mass amounted to 5.5% at six days of the fermentation, which was the maximum relative value during the fermentation. To the authors' best knowledge, this is the first report on ensiling rice straw with a selected starter at low temperature and investigation of the fermented characteristics.     

Tree growth and its association with climate between individual tree-ring series at three mountain ranges in north central China

Individual tree-ring series may show changed growth trends and divergent climate–growth associations even within a site, highlighting the need to examine tree growth and its climate association before building a chronology. We provided a case study for the stratification and temporal variability of tree growth and its climate associations of individual cores for three mountain ranges in north central China. Tree growth is mainly limited by moisture conditions in previous July–September and current June–August. Repeated sampling and field investigations of Picea wilsonii at Xinglong Mountain over a growth year of 2004 suggested that the growing season is from about the end of April to the end of September. It appears that the moisture conditions in previous and current growing seasons are crucial for tree growth in this region. However, a decrease in drought limitation was observed for a few tree-ring series. We thereby built the pooled chronology and sub-site chronologies with only drought-sensitive tree rings similar climate–growth relationships from the three mountain slopes. Growth disturbances of tree-ring series are detected by checking the occurrence of successively low values of the biweight series, which are treated by fitting a flexible curve.     

西藏高原田间冬小麦旗叶光合作用研究

 西藏高原冬小麦旗叶光合速率日变化曲线为平坦或单峰型,没有明显“午睡”现象。净光合速率日最高值可与平原接近。光合日总量最高值出现在灌浆中期,其值比平原低4％～34％。净光合速率达20μmolCO2·m-2·s-1以上的环境因子组合是光合有效辐射光量子通量密度2000μmol·m-2·s-1以上,气温25～29℃,近地层大气CO2密度0.41mg·dm-3以上,0cm地温18～23℃、5cm地温15～19℃。这样的因子组合在高原同时满足的机率不高,由于CO2浓度与光温因子高值出现时间不同步,更由于CO2密度比内陆平原低1/3,严重制约了光合日总量值,高原冬小麦旗叶光合作用的特点是净光合速率日最高值可与平原接近,但光合日总量却明显低于平原。     

Correlation between Activation of PI3K/AKT/mTOR Pathway and Prognosis of Breast Cancer in Chinese Women

Background

Abnormal activation of PI3K/AKT/mTOR (PAM) pathway, caused by PIK3CA mutation, KRAS mutation, PTEN loss, or AKT1 mutation, is one of the most frequent signaling abnormalities in breast carcinoma. However, distribution and frequencies of mutations in PAM pathway are unclear in breast cancer patients from the mainland of China and the correlation between these mutations and breast cancer outcome remains to be identified.

Methods

A total of 288 patients with invasive ductal breast cancer were recruited in this study. Mutations in PIK3CA (exons 4, 9 and 20), KRAS (exon 2) and AKT1 (exon 3) were detected using Sanger sequencing. PTEN loss was measured by immunohistochemistry assay. Correlations between these genetic aberrations and clinicopathological features were analyzed.

Results

The frequencies of PIK3CA mutation, KRAS mutation, AKT1 mutation and PTEN loss were 15.6%, 1.8%, 4.4% and 35.3%, respectively. However, except for PTEN loss, which was tied to estrogen receptor (ER) status, these alterations were not associated with other clinicopathological features. Survival analysis demonstrated that PIK3CA mutation, PTEN loss and PAM pathway activation were not associated with disease-free survival (DFS). Subgroup analysis of patients with ER positive tumors revealed that PIK3CA mutation more strongly reduced DFS compared to wild-type PIK3CA (76.2% vs. 54.2%; P = 0.011). PIK3CA mutation was also an independent factor for bad prognosis in ER positive patients.

Conclusions

AKT1, KRAS and PIK3CA mutations and PTEN loss all exist in women with breast cancer in the mainland China. PIK3CA mutation may contribute to the poor outcome of ER positive breast carcinomas, providing evidence for the combination of PI3K/AKT/mTOR inhibitors and endocrine therapy.