Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1

The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the km was 2.6 × 10-8 m and the kcat was 1.6 × 10-2 sec-1. For linear E. histolytica DNA substrate the Km and kcat values were 1.3 × 10-8 m and 2.2 × 10-4 sec-1 respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg2+ was required for activity, 60% activity was seen with Mn2+ and none with other divalent metal ions. Substitution of PDX12-14D (a metal-binding motif) with PAX12-14D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome

Labeling of Unique Sequences in Double-Stranded DNA at Sites of Vicinal Nicks Generated by Nicking Endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.Wallace A. Coulter Foundatio

Investigating enzyme communication during base excision repair in Escherichia coli

Mismatch uracil DNA Glycosylase (MUG) from Escherichia coli is an initiating enzyme in the base excision repair (BER) pathway and is responsible for the removal of 3,N4-ethenocytosine and uracil from DNA during the stationary phase of E.coli cell growth. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. MUG is widely regarded as a “single turnover” enzyme because it still remains tightly bound to its abasic product after cleavage, thus impeding its catalytic turnover. This may be a general protective mechanism to protect the abasic BER intermediate, whereby coordination of enzyme activity in BER is achieved through displacement of the DNA glycosylase by the downstream apurinic-apyrimidinic (AP) endonuclease. Numerous DNA glycosylases have now been cited as having an enhanced turnover in the presence of an AP endonuclease. The aim of this project is to investigate enzyme coordination between MUG and its both downstream AP endonucleases, Exonuclease III (ExoIII) and Endonuclease IV (EndoIV), in the initial steps of BER. We show here that MUG binds its substrate, abasic DNA and non-specific DNA in the differential modes. A 2:1 cooperative binding stoichiometry with abasic DNA is demonstrated to be of functional significance in both product binding and catalysis via fluorescence anisotropy assays, band shift assays and loss-of-function site-directed mutagenesis methods. The effects of the ExoIII and EndoIV on the MUG turnover kinetics with a U•G containing substrate was investigated. Both ExoIII and EndoIV greatly enhance the turnover of MUG. Furthermore, the analysis of both ExoIII catalytic activity dependent and concentration dependent on MUG turnover demonstrate ExoIII may employ a product scavenging mechanism to enhance MUG turnover. These combined results constitute a new concept that MUG has a pre-catalytic discrimination ability to coordinate its reactivity behavior with the other enzymes.Open Acces

Mechanistic Studies of Specific DNA Cleavage By PvuII Restriction Endonuclease

PvuII restriction endonuclease is a homodimeric protein which recognizes and cleaves the palindromic sequence (CAG?CTG) in the presence of Mg(II) ions. Starting with PvuII as a model system, pKa calculations with crystallographically defined metal ligated water are applied to PD?D/ExK motif metallonucleases in order to investigate the activation of nucleophile in metal dependent DNA hydrolysis. These results establish the electrostatic contributions of the metal ions and the conserved Lys in lowering water pKa. The calculated pKa values of metal ligands have been used to simulate the pH dependence of Mg(II) binding to PvuII. The bell shaped pH-rate profile is dissected into three ionizations. One is recognized as from the metal ligands, and the other two have pKa?s similar to calculated metal ligated water pKa in the absence of DNA. The determined pH profiles agree well with previous pH dependence studies on metallonucleases, and the correlation with pKa calculations indicates the direct involvement of metal activated water in catalysis. The different metal occupancies observed in crystal structures lead to controversy regarding the number and function of metal ions involved in DNA hydrolysis by type II restriction endonucleases. Quench flow experiments are used to monitor Mg(II) dependent single and multiple turnover DNA cleavage reactions with PvuII. Several models which differ in order of binding and the number of metal ions supporting catalysis are examined by global fits using DynaFit. The best fitted model has a preference of binding order in the reaction scheme and supports one-metal ion catalysis with 50 fold reduced activity compared with two-metal ion catalysis. The same model is also found to account for multiple turnover data in fits and simulations. A unique reaction scheme for PvuII is established to interpret the determined Mg(II) dependence of kinetic data, which provides an insight into Mg(II) participation in substrate binding, catalysis and product dissociation by restriction endonucleases

DEMETER DNA 탈메틸 효소의 모듈형 구성 개발과 후성유전체 편집 기술을 위한 응용 연구

학위논문(박사)--서울대학교 대학원 :농업생명과학대학 식물생산과학부(원예과학전공),2019. 8. 허진회.DNA methylation is a key epigenetic modification which regulates gene expression and chromatin structure in higher eukaryotes. DNA methylation is often mitotically heritable but can be removed by active mechanisms in an enzyme-dependent manner. In Arabidopsis, the DEMETER (DME) DNA glycosylase specifically excises 5-methylcytosine (5mC), generating harmful 3′ blocking intermediates, which should be immediately processed by subsequent base excision repair machineries. DME and three other family members, REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2) and DML3 share similar domain structures, but display distinct catalytic efficiencies. The DME family proteins are large, multi-domain DNA glycosylases with variable sequences connecting conserved domains, but the contribution of these structures to the regulation of catalytic activity and the enzymes required for downstream demethylation pathway after 5mC excision are largely unknown. In this study, I extensively manipulated DME protein by reducing in size to identify the minimal regions necessary for 5mC excision activity. Domain swapping experiments revealed that the glycosylase domains of DME family members are functionally equivalent, and compatibility between conserved domains is critical for DNA demethylation in vitro. In addition, I demonstrated that ABASIC ENDONUCLEASE 1-LIKE (APE1L) and ABASIC ENDONUCLEASE-REDOX PROTEIN (ARP) are responsible for trimming unusual 3′ end structures after 5mC excision, which proposes more complete DNA demethylation pathway in plants. Finally, I applied DME to epigenome editing by fusion with a transcription activator-like effector (TALE) DNA binding module. TALE-DME fusion protein showed delicate modulation of DNA methylation at specified genomic loci. Taken together, these studies will broaden our understanding of the fundamental regulatory mechanism of DNA methylation and transcription, and provide a promising avenue to produce various epigenetic traits by targeted DNA demethylation.DNA 메틸화는 중요한 후성유전학적 표지로, 고등생물에서 염색질의 구조와 유전자 발현을 조절한다. 일반적으로 DNA 메틸화는 세포 분열 이 후에도 유지가 되지만 필요한 경우 특정 효소에 의해 제거될 수 있고, 이를 DNA 탈메틸화라고 한다. 애기장대의 DEMETER (DME) DNA 글리코실라제는 DNA에 존재하는 5-메틸시토신(5mC) 염기를 특이적으로 인지하여 제거하고 생물체에 해로운 방해생성물을 만들어내는데, 이는 AP 엔도뉴클리아제 효소에 의해 안전하게 제거될 수 있다. 이 후, DNA 중합효소가 메틸기가 없는 시토신을 DNA에 넣어주게 되면 비로소 DNA 탈메틸화가 완성된다. DME 유전자군에 속하는 4개의 유전자인 DME, REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2) 그리고 DML3는 모두 비슷한 도메인 구조를 가졌지만, 서로 다른 5mC 제거 효율을 보인다. DME 유전자군에 속하는 단백질들은 크기가 크고 진화적으로 잘 보존된 여러 개의 도메인으로 이루어져 있으며, 이 도메인들은 변이가 많은 시퀀스를 통해 연결되어 있다. 하지만 이러한 구조적 특징이 DME의 효소 활성 조절에 미치는 영향이나, DME의 5mC 제거 반응 이후에 일어나는 DNA 탈메틸화 하위 경로에 대한 연구는 많이 미흡한 실정이다. 본 연구에서는 DME 단백질의 크기를 극단적으로 줄여서 5mC 제거 활성에 필요한 최소한의 도메인을 탐색하고자 했고, DNA 탈메틸 효소의 모듈형 구성 개발에 성공하였다. 이 후, 도메인 치환 실험을 통해, DME 유전자군의 글리코실라제 도메인들이 모두 비슷한 잠재적 5mC 제거 능력을 가지고 있고, 도메인들 사이의 호환성이 DME의 효소 활성에 매우 중요하게 작용한다는 것을 밝혔다. 또한, 염기 절단 수리를 담당하는 애기장대 AP 엔도뉴클리아제에 속하는 ABASIC ENDONUCLEASE 1-LIKE (APE1L)과 ABASIC ENDONUCLEASE-REDOX PROTEIN (ARP)가 DME의 효소 활성 반응 이후 생성되는 비정상적인 방해생성물을 안전하게 제거할 수 있다는 것을 증명했고, 이를 통해 식물 내 DNA 탈메틸화 경로에 대한 더욱 완성된 모델을 제시할 수 있었다. 마지막으로 DME를 후성유전체 편집 기술에 응용하기 위해 DNA 부착 모듈인 transcription activator-like effector (TALE)와 융합하는 연구를 진행하였는데, 이 과정에서 만들어진 TALE-DME 융합 단백질은 원하는 유전체 부위에서 정교하게 DNA 메틸화를 조절하는 것을 확인할 수 있었다. 종합하면, 본 연구는 DNA 탈메틸 효소의 모듈형 구성을 개발하여 도메인 기능 연구와 DNA탈메틸화 경로 연구를 위한 기초지식을 제공하였고, 이를 후성유전체 편집 기술에 응용하여 농업적으로 유용한 다양한 후성유전학적 형질 창출에 기여할 수 있을 것으로 기대된다.GENERAL INTRODUCTION 1 CHAPTER 1. Biochemical study of the domain structure of DNA demethylase 17 ABSTRACT 18 INTRODUCTION 20 MATERIALS AND METHODS 23 RESULTS 35 Three conserved domains of DME comprise the minimal entity for 5mC excision in vitro 35 Conserved domains of DME family proteins are catalytically compatible in vitro 40 Functional motifs in the conserved domains of DME 49 DISCUSSION 52 REFERENCES 58 CHAPTER 2. Biochemical study of the DNA demethylation pathway 63 ABSTRACT 64 INTRODUCTION 65 MATERIALS AND METHODS 69 RESULTS 78 DME and ROS1 5mC DNA glycosylases generate a 3′-PUA as a primary blocking intermediate in early 5mC excision 78 Enzyme-dependent δ-elimination process following 5mC excision 80 Arabidopsis encodes three putative AP endonucleases APE1L, APE2 and ARP 85 APE1L and ARP process major 5mC excision intermediate 3′-PUA to generate 3′-OH 86 3′ phosphatase activity of ARP 90 Biochemical activities of MBP-free AP endonucleases 94 ARP has AP site incision activity 94 DNA binding activity of AP endonucleases 98 DISCUSSION 100 REFERENCES 108 CHAPTER 3. Application to epigenome editing by targeted DNA demethylation 114 ABSTRACT 115 INTRODUCTION 117 MATERIALS AND METHODS 121 RESULTS 130 Generation of transgenic plants expressing TALE-DME fusion proteins 130 DNA demethylation activity of TALE-DME in transgenic plants 132 DISCUSSION 140 REFERENCES 143 ABSTRACT IN KOREAN 148Docto

Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition

The structure-specific endonuclease activity of the human XPF–ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF–ERCC1 cleaves DNA structures such as stem–loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem–loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF–ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3′-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix–hairpin–helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF–HLD shows a preference for binding stem–loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF–ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity

Distinct conformational stability and functional activity of four highly homologous endonuclease colicins

The family of conserved colicin DNases E2, E7, E8, and E9 are microbial toxins that kill bacteria through random degradation of the chromosomal DNA. In the present work, we compare side by side the conformational stabilities of these four highly homologous colicin DNases. Our results indicate that the apo-forms of these colicins are at room temperature and neutral pH in a dynamic conformational equilibrium between at least two quite distinct conformers. We show that the thermal stabilities of the apo-proteins differ by up to 20degreesC. The observed differences correlate with the observed conformational behavior, that is, the tendency of the protein to form either an open, less stable or closed, more stable conformation in solution, as deduced by both tryptophan accessibility studies and electrospray ionization mass spectrometry. Given these surprising structural differences, we next probed the catalytic activity of the four DNases and also observed a significant variation in relative activities. However, no unequivocal link between the activity of the protein and its thermal and structural stability could easily be made. The observed differences in conformational and functional properties of the four colicin DNases are surprising given that they are a closely related ( greater than or equal to65% identity) family of enzymes containing a highly conserved (betabetaalpha-Me) active site motif. The different behavior of the apo-enzymes must therefore most likely depend on more subtle changes in amino acid sequences, most likely in the exosite region (residues 72-98) that is required for specific high-affinity binding of the cognate immunity protein

The cap-snatching SFTSV endonuclease domain is an antiviral target

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (I