Phase shift and locking of RecA nucleoprotein filament
- 발행기관 서강대학교 일반대학원
- 지도교수 김도석
- 발행년도 2013
- 학위수여년월 2013. 2
- 학위명 박사
- 학과 및 전공 일반대학원 바이오융합기술협동과정
- 실제URI http://www.dcollection.net/handler/sogang/000000049459
- 본문언어 영어
- 저작권 서강대학교 논문은 저작권 보호를 받습니다.
초록/요약
RecA protein lies at the core of DNA repair mechanism in bacteria. RecA forms a helically structured filament on a single-stranded DNA (ssDNA) intermediate of homologous recombination. Filaments formed on single stranded DNA must maintain the active, stretched conformation needed to scan DNA for homology search, yet need to quickly disassemble upon completion of strand exchange. ATP hydrolysis is critical in these processes and indeed RecA everywhere in the filament continuously hydrolyzes ATP. Little is known about the time scale and the temporal ordering of conversion between different filament conformations during ATP hydrolysis. In addition, as a RecA monomer within a filament binds to three bases of the substrate DNA, there are three possible ways (phases) in the triplet selection, yet its detailed molecular mechanism has not been explored. In this thesis, I used single-molecule fluorescence resonance energy transfer (smFRET) techniques to define, for the first time, the kinetic rates with which a RecA monomer progresses through the ATP hydrolysis cycle. I found that formation of the ADP-Pi intermediate, not Pi or ADP release, licenses a RecA monomer to dissociate from the filament ends. In the middle of the filament, the cooperativity between neighboring monomers keeps the filament stable by providing a time window during which ATP exchange can restore the filament to its active state. Furthermore, the RecA filament changes its phase upon the hydrolysis of ATP. The phase shift dynamics consisted of two kinetic processes. The filaments were arrested in a specific phase by insertion of TGG repeats to the substrate DNA.
more초록/요약
RecA protein lies at the core of DNA repair mechanism in bacteria. RecA forms a helically structured filament on a single-stranded DNA (ssDNA) intermediate of homologous recombination. Filaments formed on single stranded DNA must maintain the active, stretched conformation needed to scan DNA for homology search, yet need to quickly disassemble upon completion of strand exchange. ATP hydrolysis is critical in these processes and indeed RecA everywhere in the filament continuously hydrolyzes ATP. Little is known about the time scale and the temporal ordering of conversion between different filament conformations during ATP hydrolysis. In addition, as a RecA monomer within a filament binds to three bases of the substrate DNA, there are three possible ways (phases) in the triplet selection, yet its detailed molecular mechanism has not been explored. In this thesis, I used single-molecule fluorescence resonance energy transfer (smFRET) techniques to define, for the first time, the kinetic rates with which a RecA monomer progresses through the ATP hydrolysis cycle. I found that formation of the ADP-Pi intermediate, not Pi or ADP release, licenses a RecA monomer to dissociate from the filament ends. In the middle of the filament, the cooperativity between neighboring monomers keeps the filament stable by providing a time window during which ATP exchange can restore the filament to its active state. Furthermore, the RecA filament changes its phase upon the hydrolysis of ATP. The phase shift dynamics consisted of two kinetic processes. The filaments were arrested in a specific phase by insertion of TGG repeats to the substrate DNA.
more