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Chapter 22 : Transcriptional Responses to DNA Damage
Category: Microbial Genetics and Molecular Biology
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This chapter discusses the regulation of eukaryotic genes in response to DNA-damaging agents. The yeast Saccharomyces cerevisiae has been fruitfully used as a model organism to explore pathways of regulation of eukaryotic gene expression in response to DNA damage. The transcriptional responses of S. cerevisiae to DNA damaging agents are discussed in considerable detail to highlight, in a simple model system, the experimental approaches, concepts, and open questions encountered when multicellular eukaryotes are considered. Mammalian genes that are activated at the transcriptional level in response to treatment with DNA-damaging agents include some that are involved in DNA repair and repair-associated processes. However, many also encode transcription factors, secreted growth factors and growth factor receptors, protective cytoplasmic enzymes, and proteins normally associated with tissue injury and inflammation. Several of these are known or suspected proto-oncogenes. Compilations of vertebrate genes inducible by DNA-damaging agents that were characterized prior to 1995 can be found in the literature. Although now incomplete and supplemented by gene array studies, those lists still usefully illustrate the wide variety of DNA damage-responsive genes. Delineating a defined number of regulons in multicellular eukaryotes is even less feasible than in yeast. The chapter discusses some selected observations and concentrates on the elucidation of mechanisms and pathways wherever possible, and also focuses on the effects of UV radiation and IR.
Transcriptional control of RNR gene expression in S. cerevisiae. In response to DNA damage or replicational stress (Pol2 inhibition), checkpoint kinases Mec1, Rad53, and Dun1 are activated and phosphorylate the Crt1 repressor, which loses affinity for its binding sequence. The signal transduction pathway originates from DNA structure sensors such as the 9-1-1 complex (formed in S. cerevisiae from the proteins Ddc1, Rad17, and Mec3). In S phase, these sensors may be replaced by DNA polymerase ε (Pol2).
Repressor control of S. cerevisiae PHR1. Deletion of a 39-bp fragment containing a potential repressor sequence (gold box) from the PHR1 promoter results in higher constitutive expression of a PHR1-lacZ fusion gene, measured by β-galactosidase activity and in lower induction ratios after treatment with UV radiation or MMS (compare rows A and B). However, the gene is still inducible, which indicates the existence of further elements which regulate PHR1 expression. Insertion of the same 39-bp fragment into a heterologous promoter (CYC1) distal to the normal CYC1 UAS (grey boxes) results in repression of constitutive expression and UV inducibility (compare rows C and D). (Adapted from reference 221 .)
Increased protein binding to a radioactively labeled fragment containing the regulatory sequence DRE1 of the RAD2 promoter is observed in electrophoretic mobility shift assays with cell extracts prepared at different times after treatment of the cells with 4-NQO. Complexes formed are compared to extracts from untreated cells. Equal amounts of total protein were loaded per lane. (Adapted from reference 228 .)
Preirradiation accelerates the removal of UV-induced CPD from transcriptionally active and silent S. cerevisiae genes. Removal of CPD from the transcriptionally active mating-type locus MAT (dashed lines) and the silent mating-type locus HML (solid lines) was measured by using the dimer-specific T4 endonuclease and gene-specific probes. The kinetics of CPD removal after irradiation with a single UV dose of 70 J/m2 (black lines) and after irradiation with an initial dose of 25 J/m2, followed by 70 J/m2 after 1 h of incubation in growth medium (gold lines), are compared. (Adapted from reference 260 .)
Classification of MMS-regulated S. cerevisiae genes. (Data from reference 114 .)
Cluster analysis of MMS-regulated S. cerevisiae transcripts. The expression patterns for 2,610 genes whose transcript level changes threefold or more across 26 exposure conditions are visualized and lined up horizontally. The conditions used include the treatment of cells in different cell cycle stages and the use of different DNA-damaging agents. Red and green fluorescent signals (in the original) indicate decreased or increased expression levels compared to untreated controls, respectively; groups of transcripts marked by brackets on the left show primarily decreased expression. The genes (horizontal lines) are grouped according to similarities among multiple exposure conditions. For example, the transcripts in group 8 show primarily increased levels following treatment with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and not with MMS. By this analysis, 18 clusters of possibly coregulated genes are revealed. (Adapted from reference 114 .)
Transcription-independent NER is deficient in cells with a p53 defect. (A) Removal of UV radiation-induced CPD (10 J/m2) from the genome overall is detected over time by using alkaline gradient centrifugation of labeled DNA following CPD-specific cleavage with T4 endonuclease (endo) V in wild-type fibroblasts. (B) This repair process is defective in Li-Fraumeni fibroblasts. (C) In the wild type, the same process is analyzed for the DHFR gene with strand-specific probes. (D) Li-Fraumeni cells show a specific defect in CPD removal from the nontranscribed strand. (Adapted from reference 74 .)
A cascade of protein kinases and other activators originating from the cell membrane results in activation of the transcription factors AP-1 following UV radiation. Note that transcription of JUN is activated by an AP-1-like transcription factor containing ATF-2 and JUN itself.
In response to growth factors, the small GTP-binding protein RAS is activated and binds to RAF-1 (a MAPK kinase kinase). This binding results in phosphorylation of MEK (a MAPK kinase) and activation of a MAPK subpathway. (Adapted from reference 45 .)
Effect of UV-B irradiation on aggregation and internalization of EGF and TNF receptors (EGF-R, TNF-R) in the presence of the corresponding ligands. (Adapted from reference 205 .)
Dose response of transcript inducibility in NER-deficient human cells. Compared to wild-type cells, collagenase I and metallothionein transcripts are induced at lower doses of UV radiation in XP-A and CS-A cells but not in XP-C cells. Unrepaired photoproducts in transcribed but not in nontranscribed DNA appear to sensitize the UV response. (Adapted from reference 22 .)
The capacity for double-strand break repair in nondividing human cells increases with the IR dose. Confluent primary human lung fibroblasts (MRC-5 cells) were treated with the dose of X rays indicated, and foci of γ-H2AX per cell as a measure for unrepaired double-strand breaks were counted during the period of postirradiation incubation. The gold areas of the bars represent the endogenous double-strand break background. At a very low double-strand break frequency, essentially no repair is detectable. (Adapted from reference 207 .)