효모의 새로운 유전자 HRQ1과 CMR1의 생물학적 기능에 대한 연구 = Funtional studies of novle genes HQR1 and CMR1 in Saccharomyces cerevisiae

다국어 초록 (Multilingual Abstract)

Abstract

Maintenance of genome integrity involves a number of conserved proteins, including RecQ helicases, which play multiple roles at various steps in homologous recombination and DNA repair pathways. Sgs1 has been described as the only RecQ helicase in lower eukaryotes. However, recent studies revealed the second RecQ helicase, Hrq1, which is most similar to human RECQL4. Here, I show that hrq1Δ mutation resulted in increased mitotic recombination and spontaneous mutation in Saccharomyces cerevisiae. I also observe that hrq1Δ was sensitive to 4-NQO and cisplatin, which was not complemented by overexpression of Sgs1. In addition, the hrq1Δsgs1Δ double mutant display synthetic growth defect as well as shortened chronological life span compared with the respective single mutants. Analysis of type of age-dependent Canr mutations revealed that only point mutations were found in hrq1Δ mutation, whereas significant gross deletions were found in sgs1Δ. The hrq1Δ mutant strain showed shortened replicative life. However, the rDNA recombination frequency and telomere length were not significantly different from those of with a wild-type strain. Mutation in genes of nucleotide excision repair pathway, but not homologous recombination repair and post replication repair pathways were found to be epistatic to hrq1Δ on 4-NQO and cisplatin induced DNA damage.
In conclusion, these results suggest that Hrq1 is most likely to be involved in recombination and DNA repair pathways in S. cerevisiae independent of Sgs1.

다국어 초록 (Multilingual Abstract)

Abstract

DNA metabolic processes such as DNA replication, recombination and repair are fundamentally important for the maintenance of genome integrity and cell viability. Although a large number of proteins involved in these pathways have been extensively studied, many proteins still remain to be identified. In this study I isolate the DNA binding proteins from Saccharomyces cerevisiae using DNA-cellulose columns. By analyzing mass spectrometry, an uncharacterized protein Cmr1/YDL156W, was identified. Cmr1 showed sequence homology to human Damaged-DNA binding protein 2 in its C terminal WD40 repeats. Consistent with this finding, purified recombinant Cmr1 was found to be intrinsically associated with DNA binding activity and exhibited higher activity with UV-damaged and distorted DNA substrates. Chromatin isolation experiment reveals that Cmr1 localized in both the chromatin fraction and supernatant fractions, and the level of Cmr1 in chromatin fraction is increased when yeast cells were irradiated with UV and exposed to 4-NQO. These results suggest that Cmr1 may be involved in DNA damage responses in yeast.

목차 (Table of Contents)

• Part I Genetic Characterization of HRQ1 1
• Abstract 2
• Introduction 4
• Materials and Methods 13
• Yeast strains and plasmids 13
• Measurement of recombination and spontaneous mutation frequencies 16
• Measurements of sensitivity to chemical agent and UV-irradiation 18
• Measurement of CLS and age-dependent mutation frequency 19
• CAN1 sequencing 19
• Measurement of RLS 20
• Telomere length analysis 21
• rDNA recombination rate 22
• Yeast two hybrid analysis 22
• Results 24
• The hrq1Δsgs1Δ double mutant displays synthetic growth defect and hyper-recombination phenotype 24
• Hrq1 and Sgs1 function in parallel pathways for regulation of CLS and maintenance of genome stability 27
• Hrq1 promote RLS 32
• The hrq1Δ mutant is sensitive to DNA damages induced by 4-NQO and cisplatin 35
• Multi-copy expression of Sgs1 does not complement 4-NQO and cisplatin sensitive phenotype of hrq1Δ mutant 38
• Hrq1 functions in NER pathway 41
• The double mutation, hrq1Δrad26Δ, hrq1Δrpb9Δ and hrq1Δrad7Δ resulted in synthetic growth defect in the presence of 4-NQO and cisplatin 44
• Dominant negative effect of hrq1-K318A is dependent on RAD4, but not RAD10 47
• Discussion 54
• Part II Genetic and Biochemical Studies of Cmr1 60
• Abstract 61
• Introduction 62
• Material and Methods 70
• Strains and media 70
• Oligonucleotides and DNA substrate preparation 70
• Isolation of DNA binding proteins from S. cerevisiae 73
• Gel filtration analysis 74
• Cloning and purification of Cmr1 75
• Chromatin purification 76
• Measurement of chemical agent and UV sensitivity 77
• Results 78
• Identification of Cmr1 78
• Cmr1 binds to both single- and double-stranded DNA 81
• The Cmr1 protein shares amino acid sequence homologies with damaged-DNA binding protein 2, DDB2 83
• Cmr1 binds preferentially to UV-damaged DNA 85
• Cmr1 preference for specific DNA structures 90
• Cmr1 is found in the chromatin fraction 94
• Discussion 100
• Reference 104
• Abstract (Korean) 116
• 감사의 글 119
• Curriculum vitae 121