Wenyuan Han:
Studies on DNA damage response in Sulfolobus islandicus

Date: 15-01-2015    Supervisor: Qunxin She

All living organisms have to keep their genetic information intact. However, environmental stimuli and endogenous factors constantly yield various DNA lesions, which impose serious challenges for cells to maintain the stability of their genetic materials. Upon severe DNA damage, cells initiate global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce programmed cell death (PCD) upon lethal DNA damage. However, little is known about DNA damage response in Archaea. To start to address the problem, I investigated the general cellular response of Sulfolobus islandicus, a model organism of Archaea, to DNA damage agents, 4-nitroquinoline 1-oxide (NQO) and methyl methanesulfonate (MMS), and hydroxyurea (HU) that may not introduce DNA lesions directly.

Comparison of the effects of the three drugs on S. islandicus cells showed that NQO and MMS led to DNA-less cell formation, while HU did not. In addition, the DNA-less cells were featured with increased side scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA damage response, suggesting the crucial roles of DSB in triggering DNA damage response. Then, NQO-induced DNA-less formation was impaired in the culture with retarded cell cycle, suggesting that DNA replication played an important role in DNA damage response in Sulfolobus.

We also investigated the roles of RG1 (RG: reverse gyrase) in cellular response to MMS in S. islandicus. MMS induced dramatic genomic DNA fragmentation, then a gradual cellular DNA content reduction and loss of the DNA fragments and at last formation of DNA-less cells. The latter two suggested two stages of genomic DNA degradation during MMS treatment. Further, the gradual cellular DNA content reduction was accompanied with degradation of RG1 (RG: reverse gyrase) during MMS treatment. In the cells with a downregulated RG1 level, MMS-induced genomic DNA degradation was significantly accelerated. In addition, RG1 was located in the chromatin part in S. islandicus, in which MMS-induced genomic DNA degradation was slower than that in S. solfataricus, where RG1 mainly existed in cytoplasm. Overall, the results supported for the hypothesis that RG1 protects genomic DNA breaks from degradation by binding to the breaks.