수소화 시키고 그대로의 비정질 Gex(아래첨자x입력불가)Se1-x(아래첨자1-x입력불가) 박막 (0.2〈х〈1)을 rf-sputtering을 이용해 상온에서 rf power 240W Ar 압력 3-6 Torr 로 제조하였다. Zn-alloy 박막인 비정질 Gex(아래첨자x입력불가)Sey(아래첨자y입력불가)Znz(아래첨자z입력불가) 박막도 같은 조건에서 Zn의 조성 27% 까지 제조하였다. 박막들의 조성은 SEM-EDAX로 구하였다. 박막들의 광흡수계수는 R-T spectra 와 photo-thermal deflection spectroscpy (...
수소화 시키고 그대로의 비정질 Gex(아래첨자x입력불가)Se1-x(아래첨자1-x입력불가) 박막 (0.2〈х〈1)을 rf-sputtering을 이용해 상온에서 rf power 240W Ar 압력 3-6 Torr 로 제조하였다. Zn-alloy 박막인 비정질 Gex(아래첨자x입력불가)Sey(아래첨자y입력불가)Znz(아래첨자z입력불가) 박막도 같은 조건에서 Zn의 조성 27% 까지 제조하였다. 박막들의 조성은 SEM-EDAX로 구하였다. 박막들의 광흡수계수는 R-T spectra 와 photo-thermal deflection spectroscpy (PDS)로부터 도출하였다. optical bandgap 과 흡수계수에 관계된 다른요소들 또 굴절율 등을 분석 도출하였다. 비정질 Ge(입력불가)Se₁(입력불가) 박막의 수소화 영향을 조사하기위해 그들의 광흡수계수와 온도에 따른 직류 전기전도도를 비교분석하였다. 비정질 Ge(입력불가)Se(입력불가)Zn(입력불가) 박막의 광학적 전기적 성질을 연구하였고 Zn의 첨가에 따른 박막의 변화는 optical bandgap 과 직류전도도의 활성화 에너지를 이용해 분석하였다. Extended X-ray absorption fine structure (EXAFS) 측정을 두 종류의 박막군에 시도하였다. 박막들의 미세구조에 대한 정보들 즉, 최근접 원자거리 및 결합배위수 등을 실험자료로부터 추출하여 다각도로 분석논의 하였다. 특히 각원자의 부분적 배위수는 이론적으로 예견된 두모델, chemically ordered bond network (OBN) 과 random bond network (RBN) 구조로부터 구해진 값들과 비교분석하였다. 그결과 비정질 Ge(입력불가)Se₁(입력불가) 박막이 조성에 상관없이 전 성분에서 4-2 공유결합을 이루었으며 Zn 는 Ge을 대치하며 배위수가 4로 판명되었다. 비정질 Ge(입력불가)Se₁(입력불가) 박막의 dielectric function 도 modelling 하여 실험값과 비교분석 하였다. 우수한 일치를 얻어내었다.The bacteriophage λO protein localizes the initiation of replication to a unique sequence, ori λ through specific protein-DNA and protein-protein interactions. Conformational changes at ori λ introduced by O protein binding have been reported and their roles have been implicated in the initiation of λ DNA replication. In our studies, we focused on detailed structural basis of the formation of the O protein-ori λ complex(O-some). This work was divided into five Chapters including a general introduction given in Chapter 1, a brief review of genetic, biochemical, and structural data for understanding the mechanism of the initiation of λ DNA replication. As shown in Chapter 2, we found that the O protein exists as a dimer and demonstrated that the active DNA binding species is also a dimer. Dimerization and sequence-specific DNA recognition are specifically mediated through the amino-terminal half of O(O1-162(아래첨자 1-162입력불가)). The binding affinity of O for a single copy of its 19 bp recognition sequence was 2-3 nM. We also found that the O-some is composed of 4 dimers of O and ori λ DNA, which contains four 19 bp direct repeat recognition sites, i, e., a dimer of O is bound to each repeat(iteron). Moreover, we found that only the amino-terminal DNA binding domain is required for formation of the O-some. To investigate the structural basis for the unique properties of O protein, we generated a number of carboxy-terminal and internal and internal deletion mutants of O. Experiments with purified mutant proteins, as shown in Chapter 3, indicated that (ⅰ) the deletion mutant retaining amino acid residues 19-110 is the smallest O protein species that can both bind to DNA and form a dimer, (ⅱ) the affinities of all mutant proteins for a single iteron are almost the same, ranging from 2 to 4 nM; (ⅲ) the portion of O that is responsible for dimerization is located between amino acid residues 19 and 85; (ⅳ) the carboxy-terminal domain (O 156-299(아래첨자 156-299입력불가)) is a monomeric species that does not recognize specific DNA sequences but instead, bind non-specifically to duplex DNA; (ⅴ) the linker joining the two structural domains is not required for O function, but its coding sequence of DNA contains several recognition sites for O protein (ori λ); and (ⅵ) a deletion! m! utant missing the amino-terminal portion of the carboxyl-terminal domain is still comparably active in the in vitro λdv replication assay. In Chapter 4, the structural basis of the O protein-DNA complex was studied in detail. Hydroxy radical footprinting was employed to obtain the high resolution structural information about the contacts between the protein and the sugar-phosphate backbone of DNA. The missing nucleoside experiment allowed us to identify energetically important base moieties that may be in contact with bound O protein. Quantitation of the extent of O-mediated DNA bending indicated that O induces a relatively sharp bend in an individual recognition sequence of 85。 ±5。 . Measurement of the O-induced topological change indicated that a region of DNA or specifically ori λis wrapped around the O protein core in a left-handed fashion with a linking number change of 0.7±0.1 turn. In Chapter 5, we present direct evidence that the O protein also has the capacity to interact with single-stranded DNA, the first such interaction discovered among prokaryotic origin-binding proteins. The implication of this dual DNA binding specificity of O for the formation of the unwound structure at the A/T-rich region of ori λ is dis cussed. The addition of the λP-DnaB comple x to the O-some produces a new nucleoprotein species with a super-shift in migration. The presence of P and DnaB reduces significantly the amount of O required for binding to single-stranded DNA. Based on these results, we propose a detailed model for sequential structural changes in ori λ as a consequence of O binding to the origin of λreplication.
수소화 시키고 그대로의 비정질 Gex(아래첨자x입력불가)Se1-x(아래첨자1-x입력불가) 박막 (0.2〈х〈1)을 rf-sputtering을 이용해 상온에서 rf power 240W Ar 압력 3-6 Torr 로 제조하였다. Zn-alloy 박막인 비정질 Gex(아래첨자x입력불가)Sey(아래첨자y입력불가)Znz(아래첨자z입력불가) 박막도 같은 조건에서 Zn의 조성 27% 까지 제조하였다. 박막들의 조성은 SEM-EDAX로 구하였다. 박막들의 광흡수계수는 R-T spectra 와 photo-thermal deflection spectroscpy (PDS)로부터 도출하였다. optical bandgap 과 흡수계수에 관계된 다른요소들 또 굴절율 등을 분석 도출하였다. 비정질 Ge(입력불가)Se₁(입력불가) 박막의 수소화 영향을 조사하기위해 그들의 광흡수계수와 온도에 따른 직류 전기전도도를 비교분석하였다. 비정질 Ge(입력불가)Se(입력불가)Zn(입력불가) 박막의 광학적 전기적 성질을 연구하였고 Zn의 첨가에 따른 박막의 변화는 optical bandgap 과 직류전도도의 활성화 에너지를 이용해 분석하였다. Extended X-ray absorption fine structure (EXAFS) 측정을 두 종류의 박막군에 시도하였다. 박막들의 미세구조에 대한 정보들 즉, 최근접 원자거리 및 결합배위수 등을 실험자료로부터 추출하여 다각도로 분석논의 하였다. 특히 각원자의 부분적 배위수는 이론적으로 예견된 두모델, chemically ordered bond network (OBN) 과 random bond network (RBN) 구조로부터 구해진 값들과 비교분석하였다. 그결과 비정질 Ge(입력불가)Se₁(입력불가) 박막이 조성에 상관없이 전 성분에서 4-2 공유결합을 이루었으며 Zn 는 Ge을 대치하며 배위수가 4로 판명되었다. 비정질 Ge(입력불가)Se₁(입력불가) 박막의 dielectric function 도 modelling 하여 실험값과 비교분석 하였다. 우수한 일치를 얻어내었다.The bacteriophage λO protein localizes the initiation of replication to a unique sequence, ori λ through specific protein-DNA and protein-protein interactions. Conformational changes at ori λ introduced by O protein binding have been reported and their roles have been implicated in the initiation of λ DNA replication. In our studies, we focused on detailed structural basis of the formation of the O protein-ori λ complex(O-some). This work was divided into five Chapters including a general introduction given in Chapter 1, a brief review of genetic, biochemical, and structural data for understanding the mechanism of the initiation of λ DNA replication. As shown in Chapter 2, we found that the O protein exists as a dimer and demonstrated that the active DNA binding species is also a dimer. Dimerization and sequence-specific DNA recognition are specifically mediated through the amino-terminal half of O(O1-162(아래첨자 1-162입력불가)). The binding affinity of O for a single copy of its 19 bp recognition sequence was 2-3 nM. We also found that the O-some is composed of 4 dimers of O and ori λ DNA, which contains four 19 bp direct repeat recognition sites, i, e., a dimer of O is bound to each repeat(iteron). Moreover, we found that only the amino-terminal DNA binding domain is required for formation of the O-some. To investigate the structural basis for the unique properties of O protein, we generated a number of carboxy-terminal and internal and internal deletion mutants of O. Experiments with purified mutant proteins, as shown in Chapter 3, indicated that (ⅰ) the deletion mutant retaining amino acid residues 19-110 is the smallest O protein species that can both bind to DNA and form a dimer, (ⅱ) the affinities of all mutant proteins for a single iteron are almost the same, ranging from 2 to 4 nM; (ⅲ) the portion of O that is responsible for dimerization is located between amino acid residues 19 and 85; (ⅳ) the carboxy-terminal domain (O 156-299(아래첨자 156-299입력불가)) is a monomeric species that does not recognize specific DNA sequences but instead, bind non-specifically to duplex DNA; (ⅴ) the linker joining the two structural domains is not required for O function, but its coding sequence of DNA contains several recognition sites for O protein (ori λ); and (ⅵ) a deletion! m! utant missing the amino-terminal portion of the carboxyl-terminal domain is still comparably active in the in vitro λdv replication assay. In Chapter 4, the structural basis of the O protein-DNA complex was studied in detail. Hydroxy radical footprinting was employed to obtain the high resolution structural information about the contacts between the protein and the sugar-phosphate backbone of DNA. The missing nucleoside experiment allowed us to identify energetically important base moieties that may be in contact with bound O protein. Quantitation of the extent of O-mediated DNA bending indicated that O induces a relatively sharp bend in an individual recognition sequence of 85。 ±5。 . Measurement of the O-induced topological change indicated that a region of DNA or specifically ori λis wrapped around the O protein core in a left-handed fashion with a linking number change of 0.7±0.1 turn. In Chapter 5, we present direct evidence that the O protein also has the capacity to interact with single-stranded DNA, the first such interaction discovered among prokaryotic origin-binding proteins. The implication of this dual DNA binding specificity of O for the formation of the unwound structure at the A/T-rich region of ori λ is dis cussed. The addition of the λP-DnaB comple x to the O-some produces a new nucleoprotein species with a super-shift in migration. The presence of P and DnaB reduces significantly the amount of O required for binding to single-stranded DNA. Based on these results, we propose a detailed model for sequential structural changes in ori λ as a consequence of O binding to the origin of λreplication.
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