S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
Regulation of Rad51-mediated DNA double-strand break repair and genetic recombination
Rad51 에 의한 DNA 이중가닥 절단 수선과 유전자 재조합 기전 연구
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- embryonic stem cell; homologous recombination; Rad51; cell cycle; DNA replication; genomic integrity; meiotic recombination; Rec8; Mek1; Cdc6; phosphorylation; crossover
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. 황덕수.
- Embryonic stem cells (ESCs) are pluripotent and self-renewing cells that originate from inner cell mass of blastocyst. ESCs should have ability to divide and grow indefinitely while sustaining their pluripotency. To preserve their self-renewal ability and faithful DNA replication responsible for genomic stability, ESCs have developed powerful machineries to preserve genomic integrity distinguished from differentiated cells, but they are not fully elucidated yet. Therefore, the suppression of mutations against DNA damage in ESCs is essential for the maintenance of genomic integrity as well as cell proliferation and inheritance of genetic trait.
Homologous recombination (HR) is one of the key processes to maintain genomic integrity against DNA replication stress. Rad51 is an important protein of HR in all eukaryotes and its functions are homology search and strand invasion. Here, I investigated that Rad51 preserves G2/M transition to regulate cell cycle progression and the level of Rad51 is a reflective of high percentage of S phase in ESCs.
ESCs exhibit prominent populations of S-phase cells compared with differentiated somatic cells. Different from many somatic cells that express Rad51 protein in cell cycle-dependent manner, Rad51 in ESCs is constitutively expressed independent of each cell cycle phases and its level is extremely higher than somatic cells. Unlike its continuously elevated protein level, the formation of Rad51 foci increased as cells enter S-phase, and decreased as cells prepare their division. The foci formation tendency is consistent with γH2AX, the marker of DNA double-strand breaks (DSBs). Also, Rad51 is entirely dissociated from chromosome during mitosis. Rad51 knockdown induces the phosphorylation of Chk1, the sign for DNA damage checkpoint activation. The FACS analysis showed that the populations of G2/M phases are accumulated and BrdU incorporation is reduced in Rad51-knockdown cells. In conclusion, HR activity of Rad51 is essential to repair spontaneously occurred DSBs, which are caused by rapid and frequent DNA replication events.
Meiosis includes a complex progression of chromosomal events which results in the physical connection of homologous chromosomes. During meiosis, cohesin complexes physically hold sister chromatids together, they are required for DSB repair and faithful chromosome segregation. Rec8 is a key component of the meiotic cohesin complex, which regulates sister chromatid cohesion and recombination between homologous chromosomes. DNA physical analysis of recombination in yeast mutant strains that Rec8 phosphorylation sites were mutated to alanines reveals a general principle: Rec8 phosphorylation is required for the timely and efficient progression of recombination at DSB-to-double Holliday Junction (dHJ) transition in the stage of homologous partner choice with the first DSB end releasing. I demonstrate that Rec8 phosphorylation does not affect for the homologous partner choice but is required for latter stages of crossover (CO)-designated meiotic recombination. Further, elimination of Mek1 kinase, which impedes checkpoint activation, relieves the meiotic progress delay caused by Rec8 deletion or Rec8 phosphorylation-defective alleles. The obtained results point to a general logic for the relationship between Rec8 and Mek1 kinase that involve in recombination progression and regulatory surveillance during meiosis.