Characterization of Novel DNA-Binding Proteins = DNA 결합 단백질의 결합 특성에 관한 연구

다국어 초록 (Multilingual Abstract)

DNA-binding proteins interact with DNA in sequence-specific and non-specific ways, playing major roles in various cellular processes, including DNA structural stability, replication, transcription, repair, recombination, and packaging. In this study, we discovered some E. coli proteins with high DNA-binding potential from anion-exchange chromatography, and selected YbdF and KdsD as candidate proteins. KdsD is a D-arabinose 5-phosphate isomerase in the lipopolysaccharide biosynthetic pathway, but its DNA-binding ability has not been previously reported. YbdF is a hypothetical protein of unknown function. Potential DNA target sequences of these DNA-binding proteins were predicted using the PADA1 modeling program, and duplex DNA that did not show significant sequence similarity to the predicted sites were used as controls. The DNA-binding affinities of two proteins were measured using fluorescence spectroscopy and EMSA. Both KdsD and YbdF bind double-stranded DNA with high affinity in a sequence-nonspecific manner. Furthermore, both proteins were observed to bind ppGpp, a bacterial stress response regulatory molecule, with high affinity. This is the first report showing KdsD and YbdF as novel DNA- and ppGpp-binding proteins.

국문 초록 (Abstract)

DNA 결합 단백질은 서열 특이적 또는 비특이적으로 DNA와 상호작용하며, DNA의 구조적 안정성, 복제, 전사, 수선, 재조합, 패키징 등 다양한 세포 과정에서 핵심적인 역할을 수행한다. 본 연구에서는 음이온 교환 크로마토그래피 분석을 통해 DNA 결합 가능성이 높은 E.coli 단백질들을 탐색하였고, 이 중 YbdF와 KdsD를 후보 단백질로 선정하였다. KdsD는 지질다당류 생합성 경로에 관여하는 D-arabinose 5-phosphate isomerase로 알려져 있으나, DNA 결합 기능은 보고된 바 없다. YbdF는 기능이 알려지지 않은 가상의 단백질(hypothetical protein)이다. 두 단백질의 잠재적 DNA 타깃 서열은 PADA1 모델링 프로그램을 통해 예측하였으며, 예측 서열과 유의한 상동성을 보이지 않는 이중가닥 DNA는 대조군으로 사용하였다. 두 단백질의 DNA 결합 친화도는 형광분광법과 EMSA를 이용하여 측정하였다. 그 결과 KdsD와 YbdF 모두 서열 비특이적인 방식으로 이중가닥 DNA에 높은 친화도로 결합하는 것으로 나타났다. 또한 두 단백질 모두 세균의 스트레스 반응을 조절하는 신호 분자인 ppGpp에 대해 강한 결합 능력을 갖는 것이 관찰되었다. 본 연구는 KdsD와 YbdF가 DNA 및 ppGpp에 결합하는 새로운 단백질임을 최초로 보고하는 것이다.

목차 (Table of Contents)

• Abstract 1
• 1. Introduction 3
• 1.1. Arabinose 5-phosphate isomerase KdsD 3
• 1.2. Hypothetical protein YbdF 4
• 1.3. DNA-binding proteins (DBPs) 10
• 1.4. Intrinsic fluorescence of proteins 11
• 1.5. guanosine tetraphosphate (ppGpp) 12
• 2. Materials and Methods 17
• 2.1. Cloning of KdsD and YbdF genes 17
• 2.2. Overexpression and purification of KdsD 17
• 2.3. Overexpression and purification of YbdF 19
• 2.4. Computational modeling of DNA-binding to proteins 20
• 2.5. Dissociation constant determination by fluorescence spectroscopy 21
• 2.6. Electrophoretic Mobility Shift Assay (EMSA) 23
• 3. Results 25
• 3.1. Determination of dissociation constants for ppGpp to KdsD 25
• 3.2. Determination of dissociation constants for ppGpp to YbdF 35
• 3.3. Unusual behavior of DNA-binding proteins during chromatographies 43
• 3.4. Isolation of KdsD 45
• 3.5. Isolation of YbdF 45
• 3.6. Computational modeling of DNA binding to proteins 47
• 3.6.1. PADA1 DNA docking Model with KdsD 47
• 3.6.2. HDOCK DNA docking model with KdsD 48
• 3.6.3. PADA1 DNA docking Model with YbdF 57
• 3.6.4. HDOCK DNA docking model with YbdF 60
• 3.7. Determination of dissociation constants for DNA to KdsD 63
• 3.8. Determination of dissociation constants for DNA to YbdF 68
• 3.9. Electrophoretic Mobility Shift Assay (EMSA) of KdsD 71
• 3.10. Electrophoretic Mobility Shift Assay (EMSA) of YbdF 73
• 4. Discussion 75
• References 78
• Korean abstract 84