J. Venema

Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts.

We investigated the contribution of the global and the transcription‐coupled nucleotide excision repair pathway to the removal of structurally different DNA lesions. The repair kinetics of UV‐induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6‐4) pyrimidone photoproducts (6‐4PPs) were determined in an active and inactive gene in normal human fibroblasts and in xeroderma pigmentosum group C (XP‐C) fibroblasts. Previously we have shown that in normal human cells exposed to a UV dose of 10 J/m2 repair of CPDs takes place via two pathways: global repair and transcription‐coupled repair, the latter being responsible for accelerated repair of CPDs in the transcribed strand of active genes. So far, no clear evidence for transcription‐coupled repair of 6‐4PPs has been presented. Here we demonstrate that 6‐4PPs really form a target for transcription‐coupled repair. In XP‐C cells, exposed to 30 J/m2 and only capable of performing transcription‐coupled repair, CPDs as well as 6‐4PPs are removed selectively and with similar kinetics from the transcribed strand of the adenosine deaminase (ADA) gene. The non‐transcribed strand of the ADA gene and the inactive 754 gene are hardly repaired. In contrast to XP‐C cells, normal cells exposed to 30 J/m2 lack strand‐specific repair of both 6‐4PPs and CPDs, suggesting that transcription‐coupled repair is overruled by global repair, probably due to severe inhibition of transcription at this high UV dose. The much more rapid repair of 6‐4PPs compared with CPDs in normal cells may be related to higher affinity of the global repair system for the former lesion. In XP‐C cells the similarity of the rate of repair of both 6‐4PPs and CPDs in the transcribed strand at 30 J/m2 indicates that transcription‐coupled repair of photolesions takes place in a sequential way. Our results strongly suggest that the significance of transcription‐coupled repair for removal of lesions depends on the type of lesion and on the dose employed.

Hierarchies of DNA repair in mammalian cells : biological consequences

Mammalian cells exposed to genotoxic agents exhibit heterogeneous levels of repair of certain types of DNA damage in various genomic regions. For UV-induced cyclobutane pyrimidine dimers we propose that at least three levels of repair exist: (1) slow repair of inactive (X-chromosomal) genes, (2) fast repair of active housekeeping genes, and (3) accelerated repair of the transcribed strand of active genes. These hierarchies of repair may be related to chromosomal banding patterns as obtained by Giemsa staining. The possible consequences of defective DNA repair in one or more of these levels may be manifested in different clinical features associated with UV-sensitive human syndromes. Moreover, molecular analysis of hprt mutations reveals that mutations are primarily generated by DNA damage in the poorly repaired non-transcribed strand of the gene.

(6-4) Photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.

Non-Random Distribution of UV-Induced Repair in Higher-Order Chromatin Loops in Human Cells and its Relationship to Preferential Repair of Active Genes

The eukaryotic genome is organized into a series of loops each topologically anchored by a skeletal structure termed scaffold or nuclear matrix. Such an organization is thought to facilitate processes which occur proximal to the nuclear matrix, i.e., replication and transcription. In confluent human fibroblasts exposed to either 5 J/m2 or 30 J/m2 no evidence was found for compartmentalization of UV-induced excision repair at the nuclear matrix, i.e., lesions do not require prior attachment to the nuclear matrix to be repaired.

Heterogeneity of DNA Repair in Relation to Chromatin Structure

Analysis of repair processes in mammalian cells has largely been focused on induction and repair of DNA damage in the genome overall. In particular the repair of ultraviolet light-induced photoproducts has been intensively studied in a variety of mammalian cells and in most cases UV-induced cytotoxicity can be correlated to the extent of unscheduled DNA synthesis or removal of pyrimidine dimers from the nuclear DNA. For example variation in UV-induced cytotoxicity found both within and between the various complementation groups of the human UV-sensitive disorder xeroderma pigmentosum (XP) generally correlates with the extent of defective excision repair (Kantor and Hull 1984). However, a notable exception to this is found in nondividing XP-cells belonging to complementation group C, which are relatively resistant to the lethal effects of UV (Kantor and Hull 1984; Mayne and Lehmann 1982). Also, in a number of other cases the removal of pyrimidine dimers from the genome overall turned out to be an invalid parameter to predict UV-induced cytotoxicity. Cockayne’s syndrome (CS) is a human disorder characterized at the cellular level by an increased sensitivity to the killing effects of UV-light, but with an apparently normal capacity to perform unscheduled DNA synthesis or to remove pyrimidine dimers (Mayne and Lehmann 1982). The various rodent cell lines consistently exhibit low levels of pyrimidine dimer removal for the genome overall (Van Zeeland et al. 1981) but are equally resistant to lethal effects of UV-light as human cells which are capable of performing fast and efficient repair of pyrimidine dimers.