In eukaryotic cells, transcription coupled nucleotide excision repair (TCR) is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the transcribed strand of a gene. Rad26, the yeast homolog of the human Cockayne syndrome group B (CSB) protein, plays an important role in TCR. Spt4, a transcription elongation factor that forms a complex with Spt5, has been shown to suppress TCR in rad26⌬ cells. Here we present evidence that Spt4 indirectly suppresses Rad26-independent TCR by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with Pol II. We further found that the C-terminal repeat (CTR) domain of Spt5, which is dispensable for cell viability and is not involved in interactions with Spt4 and Pol II, plays an important role in the suppression. The Spt5 CTR is phosphorylated by the Bur kinase. Inactivation of the Bur kinase partially alleviates TCR in rad26⌬ cells. We propose that the Spt5 CTR suppresses Rad26-independent TCR by serving as a platform for assembly of a multiple protein suppressor complex that is associated with Pol II. Phosphorylation of the Spt5 CTR by the Bur kinase may facilitate the assembly of the suppressor complex. Nucleotide excision repair (NER)2 is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs) (1). NER can be grouped into two pathways: global genomic repair (GGR), which refers to repair throughout the genome, and transcription coupled repair (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand of actively transcribed genes (2). In the yeast Saccharomyces cerevisiae, Rad7, Rad16 (3), and Elc1 (4) are specifically required for GGR, but dispensable for TCR. Rad7 and Rad16 form a complex that binds specifically to UV-damaged DNA in an ATP-dependent manner and has DNA-dependent ATPase activity (5). Elc1 has been shown to be a component of a ubiquitin ligase that contains Rad7 and Rad16, and is responsible for regulating the levels of Rad4 protein in response to UV damage (6, 7). It has also been suggested that Elc1 is a component of another ubiquitin ligase complex, which contains Ela1, Cul3, and Roc1 but not Rad7 and Rad16 (8,9). The role of Elc1 in GGR may not be subsidiary to that of Rad7 and Rad16 (4).The mechanistic details of TCR are relatively well understood in Escherichia coli. The transcription repair coupling factor Mfd targets the transcribed strand for repair by recognizing a stalled RNA polymerase and actively recruiting the NER machinery to the transcription blocking lesion as it dissociates the stalled RNA polymerase (10). Conversely, the TCR mechanisms in eukaryotes appear to be extremely complicated (for reviews, see Refs. 11 and 12). In mammalian cells, Cockayne syndrome group A (CSA) and B (CSB) proteins are specifically required for TCR, but dispensable for GGR (13-16). Like its human homolog CSB, the yeast Rad26 plays an important role in TCR but is dispensable for GGR (17). Both human CSB (18) ...

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Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. Rad26, a member of the Swi2/Snf2 superfamily of proteins, has been shown to be involved in a specialized NER process called transcription coupled NER. Rad16, another member of the same protein superfamily, has been shown to be required for genome-wide NER. Here we show that Rad16 and Rad26 play different roles in repairing repressed and actively transcribed genes in yeast. Rad16 is partially dispensable, and Rad26 plays a significant role in repairing certain regions of the repressed GAL1-10, PHO5 and ADH2 genes, especially in the core DNA of well-positioned nucleosomes. Simultaneous elimination of Rad16 and Rad26 results in no detectable repair in these regions of the repressed genes. Transcriptional induction of the GAL1-10 genes abolishes the role of Rad26, but does not affect the role of Rad16 in repairing the nontranscribed strand of the genes. Interestingly, when the transcription activator Gal4 is eliminated from the cells, Rad16 becomes partially dispensable and Rad26 plays a significant role in repairing both strands of the GAL1-10 genes even under inducing conditions. Our results suggest that Rad16 and Rad26 play different and, to some extent, complementary roles in repairing both strands of repressed genes, although the relative contributions of the two proteins can be different from gene to gene, and from region to region of a gene. However, Rad16 is solely responsible for repairing the nontranscribed strand of actively transcribed genes.

Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.

There is little understanding of the effect that reactive oxygen metabolites have on cellular behavior during the processes of invasion and metastasis. These oxygen metabolites could interact with a number of targets modulating their function such as enzymes involved in basement membrane dissolution, adhesion molecules involved in motility or receptors involved in proliferation. We investigated the effect of increased scavenging of superoxide anions on the expression of the urokinase receptor (uPAR) in PC-3M human prostate cancer cells. Urokinase receptor is a GPIlinked cell surface molecule which mediates multiple functions including adhesion, proliferation and pericellular proteolysis. Addition of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) to PC-3M cultures stimulated expression of uPAR protein peaking between 48 and 72 hours. Cell surface expression of the uPAR was also increased. Surprisingly, uPAR transcript levels increased only slightly and this mild increase did not coincide with the striking degree of protein increase. This disparity indicates that the TEMPOL effect on uPAR occurs through a post-transcriptional mechanism. TEMPOL presence in PC-3M cultures reduced intracellular superoxide-type species by 75% as assayed by NBT dye conversion; however this reduction significantly diminished within hours following TEMPOL removal. The time gap between TEMPOL treatment and peak uPAR protein expression suggests that reduction of reactive oxygen metabolites in prostate cancer cells initiates a multistep pathway which requires several hours to culminate in uPAR induction. These findings reveal a novel pathway for uPAR regulation involving reactive oxygens such as superoxide anion. FindingsReactive oxygen species (ROS) are becoming increasingly associated with several aspects of cancer progression including not only carcinogenesis but also tumor cell proliferation and invasion [1]. In prostate cancer, oxygen radicals are reported to arise from several sources within the cells including the NADPH oxidase [1], mitochondrial glycerophosphate-dependent ROS [2], xanthine oxidase and nitric oxide synthases [3]. The cell's net redox state is a balance between oxygen radical synthesis and breakdown, and net ROS metabolism in prostate cancer arises via activities of the scavenger enzyme systems catalase, superoxide dismutase I (Zn2+/Cu2+ SOD) and II (MN-SOD), and glutathione peroxidase [3].