Frances Mendez

Heat Shock Protein 70 Binds to Human Apurinic/Apyrimidinic Endonuclease and Stimulates Endonuclease Activity at Abasic Sites

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.

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase β

Abstract Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase β, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase β was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase β repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase β also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.

Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells

Abstract Mendez, F., Sandigursky, M., Franklin, W.A., Kenny, M.K., Kureekattil, R. and Bases, R. Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells. Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase β (β pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150–180-kDa fractions to 30–70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein–protein associations are disrupted, but β pol was not displaced by this treatment. UDG was not essential to the presence of β pol in the 150–180-kDa enzyme complex. β pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and β pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and β pol in protein–protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150–180-kDa and 30–70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein–protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Topoisomerase inhibition by lucanthone, an adjuvant in radiation therapy

PURPOSE To determine whether lucanthone can inhibit human topoisomerases in vitro. METHODS AND MATERIALS Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. RESULTS Incubation of the enzymes with lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). CONCLUSION Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.

Protein-Protein Interactions between the Escherichia coli Single-Stranded DNA-Binding Protein and Exonuclease I

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.

DNA of HeLa Cells during Caffeine-promoted Recovery from X-ray Induced G2 Arrest

Progression of X-irradiated HeLa cells from G2 arrest through mitosis was promoted by 1mM caffeine. Caffeine promoted the return from abnormally high levels of radiation-induced immunoreactivity to antinucleoside antibodies, which indicates persistent DNA strand separation, to the low levels normally found in G2. With caffeine, the irradiated cells progressed through mitosis, producing daughter cells with the normal G1 content of DNA. Without caffeine, the DNA content of individual radiation-arrested cells retained G2 values and the abnormally high levels of immunoreactivity to antinucleoside antibodies.

Abasic Sites in DNA of HeLa Cells Induced by Lucanthone

Abasic sites in HeLa cell DNA were increased in frequency by exposing the cells to lucanthone. Cell growth in the presence of lucanthone caused progressive accumulation of abasic sites and loss of cellular DNA. After 2 hr in 8 μM lucanthone, the abundance of abasic sites was 2.4 fold greater than the background of 9.9±2.0 SE abasic sites/106 nucleotides; 80 μM lucanthone in the growth medium increased the level 12.6±2.5 SE fold and decreased the DNA content in HeLa cells to one-half of the value obtained in untreated cells. The frequency of abasic sites in cellular DNA was determined by the aldehyde reactive probe method, with reference to abasic sites created in plasmid pBR322. The ability of lucanthone to inhibit the normal repair of abasic sites might reflect inhibition of apurinic/apyrimidinic endonuclease (HAP1) by the drug, thereby preventing an early step in the base excision repair pathway. Unrepaired abasic sites prevalent after ionizing radiation are cytotoxic lesions that promote DNA strand breaks. These results suggest a rationale for the joint lethal effects of lucanthone and ionizing radiation in cells and the accelerated tumor regression observed in cancer patients who received the combined therapy.

Specific stimulation of human apurinic/apyrimidinic endonuclease by heat shock protein 70.

We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1-385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386-640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1.

Carcinogen-induced immunoreactivity to antinucleoside antibodies in HeLa cells

Abstract Immunoreactivity to fluorescein-labelled antinucleoside antibodies was induced in HeLa cell nuclei by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). DNA strand breaks induced by MNNG were detected by measuring the influence of MNNG on the sedimentation in alkaline sucrose gradients of a fast sedimenting DNA-Containing complex. Dissociation of the complex and induction of immunoreactivity both proved to be sensitive, dose-dependent indicators of MNNG action in HeLa cells. Since antibodies directed against nucleosides react only with denatured or single-stranded DNA, it appears likely that MNNG-induced immunoreactivity results from exposure of single-stranded regions of DNA, providing a means for the rapid identification of carcinogen action by immunofluorescent techniques. N-Acetoxy-2-acetylaminofluorene also induced immunoreactivity and led to the dissociation of the DNA complex in HeLa cells. Seven other chemicals with known carcinogenic properties also induced immunoreactivity in this test. Immunoreactivity and dissociation of the complex were not induced by inhibitors of DNA synthesis such as hydroxyurea and cytosine arabinoside. Protein synthesis inhibitors such as cycloheximide induced immunoreactivity but did not dissociate the complex. Cycloheximide-induced immunoreactivity was rapidly and completely reversible following removal of the drug; the effects of MNNG were not reversible in our experiments. This novel application of immunofluorescent techniques provides a rapid quantitative means for detecting and studying the action of carcinogens in animal cells.

DISSOCIATION OF HISTONE AND DNA SYNTHESIS IN X-IRRADIATED HeLa CELLS.

Abstract Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1 000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis continued with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal.