DNA repair in the MYC and FMS proto-oncogenes in ultraviolet light-irradiated human HL60 promyelocytic cells during differentiation.

In order to better understand the role of transcription in cellular processing of damage in specific DNA sequences, we have used an in vitro differentiation system to modulate the activity of the MYC gene. When human HL60 promyelocytic cells differentiate in vitro, the transcriptional activity of the MYC gene is down-regulated. We have shown that in the expressed MYC gene, 56% of UV-induced cyclobutane pyrimidine dimers (CPDs) are removed within 18 h and the transcribed strand is selectively repaired. However, late in differentiation, when the MYC gene is maximally down-regulated, only 15% of the CPDs are removed within the same period. During early differentiation, the MYC gene is regulated by a block to transcription elongation at the 5' end of the first intron. Our results reveal no significant difference in the rate of CPD removal between the restriction fragments upstream and downstream of this elongation block. Furthermore, both strands of each fragment exhibit similar repair characteristics. In contrast, the constitutively expressed FMS gene exhibits proficient removal of CPD in both the differentiated and undifferentiated cells. Furthermore, the repair appears to be more proficient at the 5' end (exon 1) than in the 3' end of the gene about 35 kilobases downstream from exon 1. Since efficient repair of the active FMS gene is maintained in the differentiated cells the loss of repair competence seen in MYC is more likely associated with its reduced transcriptional activity than with a decrease in the overall repair capacity of the terminally differentiated cells.

Differential introduction and repair of psoralen photoadducts to DNA in specific human genes.

We have developed a novel procedure to measure interstrand DNA cross-linking in specific DNA sequences. After alkaline denaturation, CsCl gradient equilibrium sedimentation at pH 10.8 is used to resolve cross-linked double-stranded DNA from un-cross-linked single-stranded DNA. The DNA in gradient fractions is slot-blotted and hybridized with 32P-labeled DNA probes for the sequences of interest. After densitometric quantitation of the autoradiograms, the fraction of DNA cross-linked is determined by the ratio of cross-linked DNA to total DNA (the sum of cross-linked and un-cross-linked DNA). We have used this approach to measure the initial levels of production and extent of repair of the photoadducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen, i.e., both interstrand cross-links and cross-linkable monoadducts, in specific DNA sequences in cultured human cells. Under conditions in which DNA fragments carrying the expressed dihydrofolate reductase gene were extensively modified, with approximately 92% of the fragments cross-linked, only 37% of the fragments containing the unexpressed fms protooncogene were cross-linked. The overall level of cross-linking for bulk DNA was 74%. Within 24 h, 90% of the cross-linking had been removed from the dihydrofolate reductase gene, whereas little removal was detected in fms, and the bulk DNA showed 31% removal. From this study, we conclude that both the introduction and removal of 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts are dependent upon the target DNA sequence and its transcriptional activity. The implications for DNA repair of chromatin structure and active transcription are discussed in relation to our results.

Transcription-coupled repair of psoralen cross-links but not monoadducts in Chinese hamster ovary cells.

We have examined the rate and extent of removal of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linkable monoadducts and interstrand cross-links from restriction fragments within the amplicon containing the dihydrofolate reductase (DHFR) gene in the Chinese hamster ovary (CHO) cell line B11. The rate and extent of removal of HMT cross-links was significantly greater in an actively transcribed fragment than in a nontranscribed extragenic fragment of similar size. For the 5' half of the DHFR gene, approximately 80% of the HMT cross-links were removed in 8 h, in agreement with results reported by Vos and Wauthier [Vos, J. M., & Wauthier, E. L. (1991) Mol. Cell Biol. 11, 2245-2252, 1991]. However, few cross-links were removed in that period from the nontranscribed fragments, whose 5' end is approximately 7 kb downstream from the DHFR transcription unit and which includes a putative replication initiation site. Even after 24 h, only about 50% of the cross-links had been removed from this fragment. In contrast, both the rate and the extent of removal of cross-linkable HMT monoadducts were similar in the two fragments with 50% of the cross-linkable monoadducts removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed in 24 h. Moreover, monoadducts formed in the bulk of the genome were removed at a slightly slower rate and to a lesser extent (30% in 24 hours) than those from either of these specific sequences.(ABSTRACT TRUNCATED AT 250 WORDS)