Summary NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention. We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NOO1 activity. Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme. Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity. Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line. Expression and purification of recombinant wild-type and mutant protein from E. coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein. PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells. Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded. The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation. These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.

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Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes [1][2][3]. This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary activity in the DNA base excision repair pathway. Although UDG activities have been shown to be present in several thermophiles [6][7][8], no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG [9]. Here, we describe a UDG from the thermophile Thermotoga maritima. The T. maritima UDG gene has a low level of homology to the E. coli G-T/U mismatch-specific DNA glycosylase gene (mug). The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C. The enzyme is also active on single-stranded DNA containing uracil. Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2‐deoxyribose‐5‐phosphate from single‐strand interruptions in DNA with a base‐free residue on the 5′ side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5′‐phosphatase activity. The enzyme has a mol. wt of approximately 50,000‐55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar‐phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10 -100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress. HSP701 is a member of a family of proteins, the transcription of which is stimulated in response to heat shock, oxidation, and other stresses in eukaryotes and prokaryotes (1, 2). Upon association, HSPs alter the folding of some proteins and often enhance their biological functions (3). In general, the chaperone functions of HSPs improve cellular responses to stress. HSP70 is an ATPase and plays a role in the transport of certain proteins within the cell (4). Although HSP70 helps protect cells against oxidative stress, it is unclear how it functions in this role (3).Base excision repair (BER) is a major repair pathway and is responsible for correcting much of the DNA damage caused by ionizing radiation and reactive oxygen species. Much of the core enzyme machinery involved in BER has been described; however, the regulation and coordination of BER with other cellular processes is not well understood. HAP1 (also known as Ape1 and Ref-1) is the first enzyme in the BER pathway that is utilized to repair most if not all of the types of damage acted on by this process. Thus, it is a good candidate for a target of regulation.We previously reported that human HSP70 could bind to HAP1 as determined by affinity chromatography and hydroxyl radical footprinting (5). Here we confirm the association using immunoprecipitation and electrophoretic mobility shift assays. HSP70 also markedly enhanced the specific endonuclease activity of HAP1. This report and our previously reported results (5) are the first indication of a role for HSPs in BER in human cells. EXPERIMENTAL PROCEDURESMaterials-Recombinant human HAP1 (His 6 -tagged) was expressed in Escherichia coli and purified to apparent homogeneity. The HAP1 expression plasmid was a kind gift from Dr. Ian Hickson (University of Oxford, United Kingdom). Purified human uracil-DNA glycosylase, UDG⌬84, a recombinant enzyme lacking 84 amino acids at the N terminus, was a generous gift from Dr. G. Slupphaug (UNIGEN, University of Trondheim, Norway). Recombinant human HSP70 and rabbit antiserum against HSP70 were purchased from StressGen (Victoria, British Columbia, Canada). Rabbit IgG against HAP1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Bovine serum albumin was obtained from Sigma. USB T4 polynucleotide kinase and [␥-32 P] A...