The abasic site (apurinic/apyrimidinic site) is the most common lesion in DNA and is suggested to be an important intermediate in mutagenesis and carcinogenesis. We have recently reported a novel assay for the detection and quantitation of abasic sites in DNA [Kubo, K., Ide, H., Wallace, S. S., & Kow, Y. W. (1992) Biochemistry 31, 3703-3708]. In this assay, the aldehyde group in an abasic site is first modified by a probe bearing a biotin residue, called the Aldehyde Reactive Probe (ARP) and then the tagged biotin is quantified by an ELISA-like assay. However, in the previous study, ARP was prepared only in a crude form, and no solid chemical data concerning the structure and specificity of ARP were reported. In this study, an improved method for the preparative synthesis of ARP has been established, and its structure has been unambiguously characterized using spectroscopic means. In order to elucidate the specificity of ARP to DNA damages, ARP was incubated with a variety of damaged bases or nucleosides and the reaction mixtures were analyzed by HPLC. Of the 14 compounds tested for their reactivity to ARP, 2-deoxyribose (a model compound for an abasic site) and 5-formyluracil reacted with ARP. Interestingly, compounds bearing a formamide group such as formamidopyrimidine and deoxyribosylformamide did not react with ARP, indicating that ARP is specific to damages having an alkyl or allyl aldehyde group. Furthermore, the ability of ARP synthesized by the defined chemical route to detect abasic sites has been substantiated using natural DNA containing abasic sites. Potential applications and limitations of the ARP assay are discussed.

The nth and nei genes of Escherichia coli affect the production of endonuclease III and endonuclease VIII, respectively, glycosylases/apurinic lyases that attack DNA damaged by oxidizing agents. Here, we provide evidence that oxidative lethal lesions are repaired by both endonuclease III and endonuclease VIII and that spontaneous mutagenic lesions are repaired mainly by endonuclease III.Endonuclease III (endo III) has an apurinic (AP) lyase activity at apurinic/apyrimidinic sites in DNA and has a DNA glycosylase activity that excises oxidative pyrimidine residues such as thymine glycol (2,5,8,11,14). Escherichia coli mutants defective in endo III (nth mutants) were found to exhibit normal resistance to DNA-damaging agents such as hydrogen peroxide and X rays (7). Since thymine glycol has been shown to be a lethal lesion in vitro (6, 12, 13) and in vivo (10,15,17), and a mutagenic lesion in vitro (4), the possibility existed that alternative pathways for the repair of these damages are present in E. coli. The availability of nth mutants facilitated the identification of a distinct enzyme with similar properties, designated endonuclease VIII (endo VIII) (16). Endo VIII releases modified pyrimidine bases and possesses DNA glycosylase and AP lyase activities. The gene coding for endo VIII was named nei.To understand the role of thymine glycol and modified pyrimidine bases in lethality and mutagenesis, it is important to determine the properties of nei and nth single mutation and nei nth double mutation. We report here the cloning and sequencing of the nei ϩ gene, coding for endo VIII, and the use of the cloned gene to construct an endo VIII-defective mutant of E. coli.Isolation of nei and nth plasmids. The structural gene for endo III (nth) of E. coli was assigned on Clark-Carbon plasmid pLC3-6, which contains DNA fragments at 36 min on the linkage map (18). Therefore, using pLC3-6, a 5-kb EcoRI-KpnI fragment including the nth gene was ligated on pTZ18R, named pTZ18Rnth. The sequence of a portion of this fragment agreed perfectly with that of the nth gene (data not shown).The DNA sequence of the structural gene coding for endo VIII (nei) of E. coli appears in the GenBank database under accession no. U38616. We have previously cloned a 20-kb DNA fragment, named pKY1, containing the structural gene coding for photolyase (phr) of E. coli, which is mapped at 16 min (22). Part of the detailed physical map of pKY1 looks like that of nei. We therefore subcloned the 5-kb PstI fragment from pKY1 to pTZ18R. The DNA sequence of the fragments completely agreed with that of U38616. The detailed restriction map is shown in Fig. 1. Thus, plasmid pTZ18Rnei carries the nei ϩ gene. This finding further shows that the location of the nei gene is about 5 kb clockwise from the phr gene.Construction of nei and nth mutants. In the 5-kb EcoRIKpnI fragment containing nth ϩ , there are two PstI sites, one within the nth gene and the other 37 bp upstream from the ATG start codon. The nth ϩ gene was inactivated by cloning a 1.5-kb chlorampheni...

The activity of human methylpurine DNA N-glycosylase (hMPG) for major substrates was directly compared using two types of substrates, i.e., natural DNA and synthetic oligonucleotides. By the use of ARP assay detecting abasic sites in DNA, we first investigated the activity on the natural DNA substrates containing methylpurines, ethenopurines, or hypoxanthine (Hx) prepared by the conventional methods. After the treatment with hMPG, the amount of AP sites in methylated DNA was much higher than that in DNA containing ethenopurines or Hx. The oligodeoxynucleotide having a single 7-methylguanine (7-mG) was newly synthesized in addition to 1, N(6)-ethenoadenine (epsilonA)-, Hx-, and 8-oxoguanine-containing oligonucleotides. 7-mG was effectively excised by hMPG, though it might be less toxic than the other methylated bases with respect to mutagenesis and cell killing. The kinetic study demonstrated that k(cat)/K(m) ratios of the enzyme for epsilonA, Hx, and 7-mG were 2.5 x 10(-3), 1.4 x 10(-3), and 4 x 10(-4) min(-1) nM(-1), respectively. The oligonucleotides containing epsilonA effectively competed against 7-mG, while Hx substrates showed unexpectedly low competition. Concerning the effect of the base opposite damage, hMPG much preferred Hx.T to other Hx pairs, and epsilonA.C and epsilonA.A pairs were better substrates than epsilonA.T.

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Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

Measles diagnosis has thus far been based on clinical symptoms in daily clinical practice. However, atypical cases are not uncommon; a simple and rapid method of measles diagnosis is clinically required. Lateral flow-based rapid diagnosis reagents are widely used for point-of-care testing in Japanese clinics, because it does not require special skills and facilities. We have developed a rapid diagnostic reagent for measles that employs the lateral flow method. The lower limit of detection of this reagent was almost the same for recombinant proteins of wild-type and vaccine strain, at 5.1 x 10(3) copies/test. This lower limit of detection and the specificity of this reagent suggest its efficacy for the diagnosis of measles infection.

Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.