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We studied the combined effects of Tat and general trans-activators, such as ElA and phorbol esters, on human immunodeficiency virus-1 (HIV-1) gene expression. Interaction between these two types of trans-activators may be involved in the transition from transcriptional quiesence during viral latency to active gene expression during productive infection. ElA cooperated with Tat to produce a fourfold greater increase in accumulation of full-length, cytoplasmic HIV-1-directed RNA than is expected if they were acting additively to increase RNA accumulation. Similarily, phorbol 12-myristate 13-acetate (PMA) also cooperated with Tat to elevate HIV RNA levels synergistically. Analysis of transcription rates across the HIV-1-directed transcription unit indicated, unexpectedly, that synergy between Tat and ElA could not be accounted for by increased promoter proximal transcription rates that were merely additive. However, Tat and ElA produced a greater than additive increase in transcription rates in the 3' end of the gene. These findings imply that synergy between Tat and ElA (or other general transcriptional activators) is due principally to stabilization of transcriptional elongation. Furthermore, the observation that Tat elicits only a small increase in promoter proximal transcription in the presence of ElA suggests that the magnitude of the effect of Tat on initiation is decreased when the basal level of transcription is increased. These findings underscore the importance of the ability of Tat to stabilize elongation, as well as to stimulate initiation, in an HIV-1-directed transcription unit.

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of +X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. +X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease Im is involved in the repair of thymine glycols in vivo. +X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease HI and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. +X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease HI or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.Ionizing radiation damages DNA primarily through freeradical reactions which may cause strand breaks, purine or pyrimidine base damages, damage to the deoxyribose moiety, and DNA-DNA or DNA-protein cross-links (23, 52). Since a large variety of DNA damages are produced by ionizing radiation, the determination of the biological consequences of an individual damage that is produced against a background of other damages has been problematic.The use of small, circular, double-stranded DNA bacteriophages, such as 4X174 and PM2, has facilitated the quantitation of X-ray-induced DNA damages and the determination of their biological consequences. Single-strand breaks are produced by X rays in a moderately high yield but appear to be fairly innocuous in repair-proficient hosts (48). Doublestrand breaks are lethal in phages but are produced in low yield (34,48). In bacteriophage PM2 that was X irradiated under oxic conditions, alkali-labile sites were responsible for about 14% of the X-ray-induced lethality, and thymine ring saturation products were responsible for about 5%. Singleand double-strand breaks together account for about 15% of the X-ray-induced lethality (34). This leaves some 66% of the X-ray-induced inactivation caused by unknown alkali-stable base damages. Thymine glycols, urea residues, and apurinic (AP) sites are good models for X-ray-and free radical-induced base and sugar damages (51). Thymine glycols are thymine ring saturation products, and urea residues are fragmentation products of thymine. Both result from the degradation of unstable thymine hydroperoxides which are produced by hydroxyl radical attack on the 5-6 double bond of thymine (47). Thymine glycol is the major stable product produced by the radiolysis of thymine nucleotides (47) and has been found in DNA that was X irra...

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