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ABSTRACT: 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.
DNA Repair 04/2003; 2(3):259-71. · 4.14 Impact Factor
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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 beta, 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 beta 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 beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta 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.
Cell Stress and Chaperones 02/2003; 8(2):153-61. · 3.01 Impact Factor
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ABSTRACT: 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 microM lucanthone, the abundance of abasic sites was 2.4 fold greater than the background of 9.9 +/- 2.0 SE abasic sites/10(6) nucleotides; 80 microM 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.
Cancer Investigation 02/2002; 20(7-8):983-91. · 1.85 Impact Factor
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ABSTRACT: Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.
Journal of Cellular Physiology 11/1969; 74(3):283 - 294. · 3.87 Impact Factor
