Transcription-coupled nucleotide excision repair and its regulation by the DNA damage checkpoint.
Doctoral thesis, UCL (University College London).
Elaborate DNA repair mechanisms have evolved, allowing cells to repair damages in their genomes. Nucleotide excision repair (NER) removes a variety of helix-distorting lesions, including those caused by ultraviolet (UV) irradiation. NER operates via two subpathways. Transcription-coupled repair (TC-NER) rapidly removes transcription-blocking lesions in the transcribed strand (TS) of active genes, and in the yeast Saccharomyces cerevisiae depends on the factors Rad26 and Rpb9. Lesions in untranscribed DNA, including the non-transcribed strand (NTS) of active genes are removed slower by global genome repair (GG-NER). Besides activating specific DNA repair systems, DNA damage also leads to a global cellular response, known as the DNA damage checkpoint (DDC). Cell-cycle progression is temporarily stopped after DNA damage to allow sufficient time for repair and prevent replication or segregation of damaged chromosomes. The DDC is a complex signal transduction cascade involving a number of protein kinases, the central players in budding yeast being Mec1 and Tel1, the homologues of human ATR and ATM, respectively. Besides inhibiting cell-cycle progression, accumulating evidence suggests that DNA repair systems are also influenced by the checkpoint. I have investigated the rates of repair of UV lesions in checkpoint deficient strains of Saccharomyces cerevisiae and found that NER is significantly inhibited on both strands of an active gene in the absence of Mec1. The effect on NTS repair seems to be caused by deficient de novo synthesis of repair factors, whereas TC-NER is influenced mainly by post-translational modification of one or more pre-existing proteins. I have characterised a checkpoint-dependent phosphorylation of Rad26, and have shown using point mutants that this phosphorylation increases the TC-NER capacity of cells, establishing a new link between NER and the checkpoint. In addition to these results about the interplay between the DDC and NER pathways, preliminary data from two unrelated projects will be presented. One was an attempt to establish a system for analysis of NER factor recruitment to an artificial, highly UV-damage-prone DNA sequence. The other focussed on the regulation of UV-induced degradation of Rpb1, the largest RNA Polymerase II (RNAPII) subunit, by the DDC.
|Title:||Transcription-coupled nucleotide excision repair and its regulation by the DNA damage checkpoint|
|Open access status:||An open access version is available from UCL Discovery|
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