The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O 6 meG:T mismatches, the purification of recombinant native human MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.
Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.In all living cells the induction of DNA damage activates an extremely complex network of events involving every cell process from DNA replication and transcription to cell division, protein synthesis and degradation, and, eventually, cell death (1). A major challenge in cell biology is elucidating how these events are regulated and connected. Archaea are helpful model systems for studying pathways of DNA transactions (replication, transcription, recombination, and repair) that are thought of as simplified and ancient versions of the eukaryal ones (reviewed in Refs. 2 and 3). However, to date the response to DNA damage has been poorly investigated in Archaea.Genome sequencing has revealed the presence of archaeal genes homologous to components of the eukaryal nucleotide excision repair pathway, which is involved in the repair of UV-induced DNA lesions (4, 5). We have shown previously that the exposure of Sulfolobus solfataricus to UV light and the intercalating agent actinomycin D elicits a DNA damage response with features essentially conserved in all three domains of life, including growth arrest and transcriptional induction of nucleotide excision repair genes. UV light and actinomycin D also regulate the genes encoding two DNA-binding proteins affecting DNA conformation, namely Sul7d and Smj12 (6). Sul7d is an abundant chromosomal protein that induces DNA bending, compaction, and negative supercoiling (7). In contrast, Smj12 induces positive supercoiling (8). Although the reasons for this regulation are currently only matters of spec...
Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression. Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) light-induced DNA damage. However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea. We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are efficiently repaired in vivo in the dark, suggesting that a NER pathway is active. DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB. The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics. Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D. Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.
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