Stephen O. Opiyo

Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.

Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors

To identify divergent seven-transmembrane receptor (7TMR) candidates from the Arabidopsis thaliana genome, multiple protein classification methods were combined, including both alignment-based and alignment-free classifiers. This resolved problems in optimally training individual classifiers using limited and divergent samples, and increased stringency for candidate proteins. We identified 394 proteins as 7TMR candidates and highlighted 54 with corresponding expression patterns for further investigation.

Interspecific Comparison of Constitutive Ash Phloem Phenolic Chemistry Reveals Compounds Unique to Manchurian Ash, a Species Resistant to Emerald Ash Borer

The emerald ash borer (Agrilus planipennis, EAB) is an invasive wood-borer indigenous to Asia and is responsible for widespread ash (Fraxinus spp.) mortality in the U.S. and Canada. Resistance and susceptibility to EAB varies among Fraxinus spp., which is a result of their co-evolutionary history with the pest. We characterized constitutive phenolic profiles and lignin levels in the phloem of green, white, black, blue, European, and Manchurian ash. Phloem was sampled twice during the growing season, coinciding with phenology of early and late instar EAB. We identified 66 metabolites that displayed a pattern of variation, which corresponded strongly with phylogeny. Previously identified lignans and lignan derivatives were confirmed to be unique to Manchurian ash, and may contribute to its high level of resistance to EAB. Other compounds that had been considered unique to Manchurian ash, including hydroxycoumarins and the phenylethanoids calceolarioside A and B, were detected in closely related, but susceptible species, and thus are unlikely to contribute to EAB resistance of Manchurian ash. The distinct phenolic profile of blue ash may contribute to its relatively high resistance to EAB.

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse.

Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.

Evolution of the Kdo2-lipid A biosynthesis in bacteria

BackgroundLipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps.ResultsIn order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria.ConclusionsThe nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

Nutritional attributes of ash (Fraxinus spp.) outer bark and phloem and their relationships to resistance against the emerald ash borer

The emerald ash borer (Agrilus planipennis Fairmaire, EAB) is an alien, invasive wood-boring insect that is responsible for killing millions of ash trees since its discovery in North America in 2002. All North American ash species (Fraxinus spp.) that EAB has encountered have shown various degrees of susceptibility, while Manchurian ash (Fraxinus mandshurica Ruprecht), which shares a co-evolutionary history with this insect, is resistant. Recent studies have looked into constitutive resistance mechanisms in Manchurian ash, concentrating on the secondary phloem, which is the feeding substrate for the insect. In addition to specialized metabolism and defense-related components, primary metabolites and nutritional summaries can also be important to understand the feeding behavior of insect herbivores. Here, we have compared the nutritional characteristics (water content, total protein, free amino acids, total soluble sugars and starch, percent carbon and nitrogen, and macro- and micronutrients) of outer bark and phloem from black, green, white and Manchurian ash to determine their relevance to resistance or susceptibility to EAB. Water content and concentrations of Al, Ba, Cu, Fe, K, Li, tryptophan and an unknown compound were found to separate black and Manchurian ash from green and white ash in a principal component analysis (PCA), confirming their phylogenetic placements into two distinct clades. The traits that distinguished Manchurian ash from black ash in the PCA were water content and concentrations of total soluble sugars, histidine, lysine, methionine, ornithine, proline, sarcosine, tyramine, tyrosol, Al, Fe, K, Na, V and an unknown compound. However, only proline, tyramine and tyrosol were significantly different, and higher, in Manchurian ash than in black ash.

Mining the Arabidopsis and rice genomes for cyclophilin protein families

Cyclophilins, which possess peptidyl-prolyl isomerase activity, are cellular targets of immunosuppressant drugs and involved in a wide variety of functions. While the Arabidopsis thaliana genome contains the largest number of cyclophilins, the number of plant cyclophilins available in databases is small compared to that of other organisms. It implies that many cyclophilins are yet to be identified in plants. In order to identify cyclophilin candidates from available plant sequence data, we examined alignment-free methods based on Partial Least Squares (PLS). PLS classifier performed better than profile hidden Markov models and PSI-BLAST in identifying cyclophilins from the Arabidopsis and rice genomes.

Mining Cytochrome b561 proteins from plant genomes

Cytochrome b561 (Cyt-b561) proteins are important for plant growth, development, and prevention of damage to plants. Because of their high sequence divergence, thorough mining of Cyt-b561 proteins from plant genomes are not easy. Currently there is only one Cyt-b561 gene found in the maize and none in the soybean genome. However, 22 have been identified in the Arabidopsis thaliana genome. We tested alignment-free protein classifiers based on partial least squares (PLS) and support vector machines to identify Cyt-b561. These classifiers performed better than profile hidden Markov models and PSI-BLAST. Using these classifiers we identified new Cyt-b561-related proteins from four plant genomes.

Genome-Wide Association Mapping of Rice Resistance Genes Against Magnaporthe oryzae Isolates from Four African Countries

Rice blast disease is emerging as a major constraint to rice production in Africa. Although a traditional gene-tagging strategy using biparental crosses can effectively identify resistance (R) genes or quantitative trait loci (QTL) against Magnaporthe oryzae, the mapping procedure required is time consuming and requires many populations to investigate the genetics of resistance. In this report, we conducted a genome-wide association study (GWAS) to rapidly map rice genes conferring resistance against eight M. oryzae isolates from four African countries. We inoculated 162 rice cultivars, which were part of the rice diversity panel 1 (RDP1) and were previously genotyped with the 44,000 single-nucleotide polymorphism (SNP) chip, with the eight isolates. The GWAS identified 31 genomic regions associated with blast resistance (RABR) in the rice genome. In addition, we used polymerase chain reaction analysis to confirm the association between the Pish gene and a major RABR on chromosome 1 that was associated with resistance to four M. oryzae isolates. Our study has demonstrated the power of GWAS for the rapid identification of rice blast R or QTL genes that are effective against African populations of M. oryzae. The identified SNP markers associated with RABR can be used in breeding for resistance against rice blast in Africa.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.