• BMB Reports
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• v.31 no.4
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• pp.405-408
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• 1998

Endonuclease-sensitive sites detected by T4 endonuclease V or UvrABC nuclease treatments were compared in the dihydrofolate reductase gene of UV-irradiated Chinese hamster ovary B-11 cells. The number of endonuclease-sensitive sites detected by T4 endonuclease V treatment followed by NaOH denaturation was twice that of formamide denaturation. Repeated treatment of damaged genomic DNA with T4 endonuclease V resulted in no further increase in the number of endonuclease-sensitive sites detected. The numbers of endonuclease-sensitive sites detected by UvrABC nuclease using each denaturation condition were similar. Sequential treatment with the two endonucleases using formamide denaturation resulted in twice the number of endonuclease-sensitive sites detected by treatment of each nuclease alone. Due to a lack of AP endonuclease activity these results suggest the presence of T4 endonuclease V-sensitive sites which could be complemented by alkaline gel separation or by UvrABC nuclease treatment.

• YAKHAK HOEJI
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• v.40 no.2
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• pp.193-201
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• 1996

In order to study the role of C-terminal aromatic region of T4 endonuclease V in the interaction with substrate DNA, NMR and Fluorescence spectrum were recorded. Analysis of flu orescence emission spectra showed that C-terminal region of T4 endonuclease V is in or very near the binding site. In the HSQC spectrum of $^{15}N$-Tyr-labeled T4 endonuclease V*DNA complex, the broadening of a peak was observed. It is presumed that this peak corresponds to one among three tyrosine residues which belong to the WYKYY segment of C-terminal region of T4 endonuclease V. Interactions of peptide fragments consisting of C-terminal residues of T4 endonuclease V with DNAs(TT-, T^T-DNA) were investigated by NMR and Fluorescence experiment. The results suggest that two peptide fragments themselves bind to DNAs and their binding pattern is not an intercalation mode.

• Biomolecules & Therapeutics
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• v.2 no.1
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• pp.28-33
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• 1994

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGGTTGGCG).d(CGCCAACCGC) with a thymine dimer incorporated has been studied by proton NMR. NOE data show that, although the local environment around the thymine dimer is altered, the gross structural changes are relatively small. T$_4$endonuclease V exhibited a conformational change on complex formation with DNA. This conformational change occurred around histidine 16 which was close to tyrosine 129 located in the aromatic segment (WYKYY) near the C-terminus. It is likely that the interaction between T$_4$endonuclease V and DNA is strong since the complex was not dissociated up to 1.6 M NaCl.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.267-267
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• 1994

In order to obtain insight into the mechanism by which DNA containing a thymine photo-dimer is recognized by the excision repair enzyme, T$_4$ endonuclease V, we have taken NMR study of this protein and its complex with oligonucleotides. The conformations of five different DNA duplexes DNA I : d(GCGGATGGCG).d(CGCCTACCGC), DNA II d(GCGGTTGGCG) .d(CGCCAACCGC), DNA III : d(GCGGT ^ TGGCG) .d(CGCCAACCGC), DNA IV d(GCGGGCGGCG).d(CGCCCGCCGC) and DNA V d(GCGGCCGGCG) . d(CGCCGGCCGC) were studied by $^1$H NMR. The NMR spectra of these five DNA duplexes in the absence of the enzyme clearly show that the formation of a thymine dimer within the DNA induces only a minor distortion in the structure, and that the overall structure of B type DNA is retained. The photo-dimer formation is found to cause a large change in chemical shifts at the GC7 base pair, which is located at the 3'-side of the thymine dimer, accompanied by the major conformational change at the thymine dimer site. The binding of a mutant T$_4$ endonuclease V (E23Q), which is unable to digest DNA containing a thymine dimer, to the DNA duplex d(GCGGT ^ TGGCG)ㆍd(CGCCAACCGC) causes a large down-field shift in the imino proton resonance of GC7. Therefore, this position is thought to be either the crucial point of the interaction wi th T$_4$ endonuclease V, or the si to of a conformational change in the DNA caused by the binding of T$_4$ endonuclease V. Usually, it is very difficult to assign NMR peaks in DNA * protein complex because of severe peak overlaps. In order to overcome these peak overlaps, we used a method of deuterium incorporation.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.137-137
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• 1993

방사선, 자외선, 화학물질 등 여러 변이원에 의해 생긴 DNA 손상의 대부분은 생체내에 존재하는 효소들에 의해 수복(repair)되어 DNA는 안정하게 유지된다. T$_4$ phage 유래의 T$_4$ endonuclease V는 자외선에 의해 DNA에 pyrimidine dimer가 생겼을때 이것을 특이적으로 절제 수복하는 효소이다. 인간의 질환인 색소성 걸피증(Xeroderma pigmentosum)은 태양광선, 특히 자외선에 의해 고빈도로 피부암을 발생한다. 이 질환은 유전적으로 DNA 수복기구에 장애가 있기 때문에 일어난다. 색소성 건피증의 배양세포에 T$_4$ endonuclease V를 도입하면 세포의 DNA 수복능력이 회복되기 때문에 인간과 phage라는 서로 멀리 떨어진 생물종에 공통의 DNA 수복기구가 존재하고 있다는 것을 알 수 있다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.183-183
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• 1996

Bacteriophage T4 endonuclease V initiates the repair of ultraviolet (UV)-induced pyrimidine dimer photoproducts in duplex DNA. The mechanism of DNA strand cleavage involves four sequential stens: linear diffusion along dsDNA, pyrimidine dimer-specific binding,l pyrimidine dimer-DNA glycosylase activity, and Af lyase activity. Although crystal structure is known for this enzyme, solution structure has not been yet known. In order to elucidate the solution structure of this enzyme NMR spectroscopy was used. As a basis for the NMR peak assignment of the protein, HSQC spectrum was obtained on the uniformly $\^$15/N-labeled T4 endonuclease V. Each amide peak of the spectrum were classified according to amino acid spin systems by interpreting the spectrum of $\^$15/N amino acid-specific labeled T4 endonuclease V. The assignment was mainly obtained from three-dimensional NMR spectra such as 3D NOESY-HMQC, 3D TOCSY-HMQC. These experiments were carried out will uniformly $\^$15/N-labeled sample. In order to assign tile resonance of backbon atom, triple-resonance theree-dimensional NMR experiments were also performed using double labeled($\^$15/N$\^$13/C) sample. 3D HNCA, HN(CO)CA, HNCO, HN(CA)HA spectra were recorded for this purpose. The results of assignments were used to interpret the interaction of this enzyme with DNA. HSQC spectrum was obtained for T4 endonuclease V with specific $\^$15/N-labeled amino acids that have been known for important residue in catalysis. By comparing the spectrum of enzyme*DNA complex with that of the enzyme, we could confirm the important role of some residues of Thr, Arg, Tyr in activity. The results of assignments were also used to predict the secondary structure by chemical shift index (CSI).

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.18-18
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• 1996

Bacteriophage T4 endonuclease V initiates the repair of ultraviolet (UV)-induced pyrimidine dimer photoproducts in duplex DNA. The mechanism of DNA strand cleavage involves four sequential steps: linear diffusion along dsDNA, pyrimidine dimer-specific binding, pyrimidine dimer-DNA glycosylase activity, and AP lyase activity. (omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.80-80
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• 1995

T4 Endonuclease V (Mw 16,000) acts as a repair enzyme for UV induced pyrimidine dimers in DNA. Many researchers have studied the biochemical characteristics of the enzyme. However the precise action mechanism of T4 endo V has not fully elucidated yet. In our laboratory NMR spectroscopy technique is being used for the structural study of T4 endo V. Because of its low temperature stability and high content of ${\alpha}$-helix, the conventional $^1$H NMR technique was inapplicable. Therefore we utilized stable isotope labeling technique and so far prepared about 10 amino acid specific labeled proteins. The HSQC spectra of amino acid specific labeled proteins will help us to interpret the triple resonance 3D, 4D data which are under processing, We also studied the behaviors of specific amino acid residues whose roles might be critical. When the enzyme labeled by $\^$15/N-Thr was mixed with the substrate oligonucleotide (semispecific -TT- sequence), one crosspeak in its HSQC spectrum was completely desappeared, which means that one of seven Thr residues is in the binding site of the enzyme with DNA, This result is well consistent with previous report that implicated the Thr 2 residue in the activity of the enzyme. Similar studies were carried on the behaviors of Arg and Tyr residues.

• Journal of Photoscience
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• v.9 no.2
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• pp.186-189
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• 2002

Many of the adverse effects of solar UV exposure appear to be directly attributable to damage to epidermal DNA. In particular, cyclobutane pyrimidine dimers (CPD) may initiate mutagenic changes as well as induce signal transduction responses that lead to a loss of skin immune surveillance and micro-destruction of skin structure. Our approach is to reverse the DNA damage using prokaryotic DNA repair enzymes delivered into skin using specially engineered liposomes. T4 endonuclease V encapsulated in liposomes (T4N5 liposome lotion) enhanced DNA repair by shifting repair of CPD from the nucleotide excision to the base excision repair pathway. Following topical application to humans, increased repair limited UV-induction of cytokines, many of which are immunosuppressive. In a recent clinical study, topical treatment of UV-irradiated human skin with T4N5 liposome lotion reduced the suppression of the nickel sulfate contact hypersensitivity response. Similarly, the photoreactivating enzyme enhances repair by directly reversing CPDs after absorbing activating light. Here also treatment of UV-irradiated human skin with photoreactivating enzyme in liposomes and photoreactivating light restored the response to the contact allergen nickel sulfate. These findings confirm in humans the observation in mice that UV induced suppression of contact hypersensitivity is caused in part by CPDs. We have tested the ability of T4N5 liposome lotion to prevent UV-induced skin cancer in patients with xeroderma pigmentosum (XP), who have an elevated incidence of skin cancer resulting from a genetic defect in DNA repair. Daily use of the lotion for one year in a group of 20 XP patients reduced the average number of actinic keratoses by 68% and basal cell cancers by 30% compared to 9 patients in the placebo control group. Delivery of DNA repair enzymes to skin is a promising new approach to photoprotection.