Genome Screening Of DNA Damage Response Genes

The genome is under constant threat of damage from exogenous agents such as chemical and physical damaging agents and endogenous processes that result in DNA lesions. DNA damage contributes to the onset of cancer, aging, immune deficiencies, and other degenerative diseases. In the face of exogenous and endogenous DNA damage, cells have evolved elaborate damage response mechanisms to maintain genome stability. These mechanisms include DNA repair, cell cycle checkpoint arrest and apoptosis. Many genes participate and play important roles in these pathways. Although many genes involved in DNA damage response have been reported in previous studies, there are still plenty of novel genes yet to be identified.In this study,55genes involved in DNA damage response were identified by systematically screening the set of3235viable Schizosaccharomyces pombe haploid gene deletion mutants with UV and five DNA-damaging agents. Among55genes,23genes were reported to be involved in DNA damage response for the first time. Furthermore,34.5%of these55genes were shown to function in cell cycle related processes by Gene Ontology analysis. Flow cytometry analysis of these55mutants revealed that38genes participated in cell cycle process directly or indirectly. The cell cycle of these38mutants got arrested under normal or DNA-damaged conditions, and these mutants could be divided into four groups based on their flow cytometry phenotypes.To further characterize the functions of the genes in DNA damage response, two mutants were picked from each group with specific flow cytometry phenotype. Eight mutants (Aash2, ASPAC3F10.17, ASPBC2A9.02, ASPAC27D7.08c, Asg/73, Ameu29, Asec65and Apabl) were all sensitive to at least two DNA-damaging agents. DNA microarray and clustering analysis of these eight mutants revealed that they could be classified into four clusters, and this classification perfectly matched their flow cytometry phenotypes. Deletion any of these eight genes resulted in differential expression of genes related to DNA replication and cell division. Real time PCR, microscopic analysis and complementary experiments, together with microarray data, revealed that SPBC2A9.02and SPAC27D7.08c might be required for efficient DNA replication initiation by genetically interacting with DNA replication initiation factors Abp1and Abp2. The deletion of SGF73, MEU29, SEC65or PAB1caused cell separation defects by influencing Sepl-Ace2transcriptional pathway, and that contributed to the diploidization in the mutants. In addition, SAGA complex subunit Sgf73and meiotic expression up-regulated protein Meu29might influence DNA replication initiation through Abp1and Abp2. Signal recognition particle subunit Sec65and protein phosphatase regulatory subunit Pabl might be involved in the DNA re-replication through DNA replication regulation protein Cdc18, Cdtl and Cdt2. These results suggested that these genes took part in DNA damage response through DNA replication and cell cycle control. In this study, we also found that many other genes participated in DNA damage response through chromatin dynamics, transcriptional regulation and DNA repair, etc. Altogether, identification of these novel genes provides a solid basis to illuminate the mechanism of DNA damage response. Alkaline P-mannanase, a hemicellulase, hydrolyzes mannan into manna-oligosaccharides under alkaline environment. Manna-oligosaccharides have great commercial values. Therefore, alkaline β-mannanase has high application values in food, pharmaceutical, feed and kraft pulp industries. In order to obtain an alkaline mannanase with high yield and bioactivity, we removed163amino acids from the C terminus of a β-mannanase in alkaliphilic Bacillus sp. N16-5(MAN493) to obtain its core catalytic domain (MAN330) for secretory expression in Kluyveromyces cicerisporus. The recombinant engineered strain for MAN330production was stable even after80generations. Zymological characterizations of the truncated β-mannanase (MAN330) and its full-length protein MAN493revealed that MAN330exhibited similar optimum pH optima, temperature optima, and substrate specificities to MAN493. However, pH stability of MAN330was about7%higher than that of MAN493over the pH range from9to11. Thermal stability of MAN330showed about10%higher than that of MAN493from60℃to80℃. Therefore, MAN330was more applicable than its full-length protein MAN493in industry. Furthermore, the optimum fermentation conditions for the production of MAN330from Kluyveromyces cicerisporus were established both in flask and in15L bioreactor, and the yield of MAN330obtained from flask and bioreactor achieved1378U/ml and3795U/ml respectively. This study will provide a promising basis for industrial production and application of alkaline β-mannanase.