Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage. One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall. In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS). The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref. 4). Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin. The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR. Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage. Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair.

Xanthobacter flavus grows autotrophically by using the Calvin cycle for the fixation of CO 2 . Only 2 of the 11 enzymes of the Calvin cycle are characteristic for this pathway; the others are also present during heterotrophic growth. The key enzymes of the Calvin cycle, phosphoribulokinase and ribulosebisphosphate carboxylase, are encoded within the cbb operon, which is transcribed only during autotrophic growth. Two additional genes are located within this operon: cbbX, encoding a protein with unknown function, and cbbF, encoding fructosebisphosphatase (27,29). The transcription of the cbb operon is positively regulated by CbbR, a LysR-type transcriptional regulator, which binds to two sites in the cbb promoter (47).During autotrophic growth, X. flavus uses two fructosebisphosphatase enzymes with distinct properties. The inducible enzyme encoded by cbbF has a high level of sedoheptulosebisphosphatase activity and is stimulated by ATP. The second constitutive fructosebisphosphatase has a low level of sedoheptulosebisphosphatase activity and is not stimulated by ATP (48). In contrast to the fructosebisphosphatase isoenzyme pair, only one phoshoglycerate kinase gene, which is not encoded within the cbb operon, is employed by X. flavus. The pgk gene is constitutively expressed, but the expression level is higher during autotrophic growth than during heterotrophic growth (26).Little is known about the transketolase (EC 2.2.1.1) and fructosebisphosphate aldolase (FBP aldolase; EC 4.1.2.13) enzymes of X. flavus. Like fructosebisphosphatase and phosphoglycerate kinase, these enzymes are involved in both heterotrophic metabolism and the fixation of CO 2 via the Calvin cycle. Two unrelated mechanistically distinct types of FBP aldolase enzymes are encountered in bacteria, archaea, and eukarya (24). Class I FBP aldolases form a covalent Schiff base between the substrate and the ␣-amino group of a lysine residue during catalysis, whereas the class II enzymes depend on a divalent cation as the electrophile in the catalytic cycle (24).X. flavus (Table 1) was grown heterotrophically on succinate (10 mM) or gluconate (10 mM) and autotrophically on methanol (0.5% [vol/vol]) at 30ЊC as described previously (22,28). The activities of transketolase and FBP aldolase were determined, according to published methods (15, 49), in cell extracts which were prepared by using a French pressure cell as described previously (27). The activity of transketolase was increased sixfold following autotrophic growth on methanol compared with that of heterotrophically grown cells. In sharp contrast, the activity of FBP aldolase (without Fe 2ϩ ) was the same for both heterotrophic and autotrophic growth. Because the activity of class II FBP aldolase is dependent on Fe 2ϩ as the electrophile (24), FeSO 4 (700 M) was included in the reaction assay. Surprisingly, Fe 2ϩ did not affect the FBP aldolase activity in the cell extracts of heterotrophically grown cells but stimulated the activity of FBP aldolase in cell extracts of autotrophically grown cells ...

The role of variation in susceptibility to DNA damage induction was studied as a determinant for cellular radiosensitivity. Comparison of the radiosensitive HX142 and radioresistant RT112 cell lines previously revealed higher susceptibility to X-ray-induced DNA damage in the sensitive cell line using non-denaturing elution, but not when using alkaline unwinding. The present data also show that no difference in the amount of initial damage is seen when pulsed-field gel electrophoresis (PFGE) or comet analysis are used for DNA damage assessment. However, using the halo assay or a modified version of PFGE in which the higher DNA architecture remained partially intact, the radiosensitive cells showed steeper dose-response curves for initial DNA damage than the radioresistant cells. Analysis of the protein composition, of DNA-nucleoid structures revealed substantial differences when isolated from HX142 or RT112 cells. From our data, it is concluded that HX142 and RT112 differ in their structural organization of chromatin. As no differences in the kinetics of DNA damage rejoining were found, it is hypothesized that the same amount of lesions have a different impact in the two cell lines in that the 'presentation' of DNA damage alters the ratio of repairable to non-repairable DNA damage.

Thermal radiosensitization is thought to result from inhibition of repair of radiation-induced DNA damage, DNA double-strand breaks in particular. Since the DNA-dependent protein kinase (DNA-PK) complex plays a major role in the nonhomologous end-joining of DSBs, it has been suggested that inactivation of this complex as a whole or of its individual subunits by heat might be involved in radiosensitization by heat. To test this hypothesis further, the ability of heat to enhance the radiosensitivity of cells proficient or deficient in either Ku80 or the DNA-PK catalytic subunit (DNA-PKcs) was investigated. In cells of two Ku80-deficient and two DNA-PKcs-deficient and double-strand break-deficient cell lines, the extent of radiosensitization by heat was not reduced compared to that in both their isogenic gene-complemented counterparts as well as to that in their parental cells. Thus radiosensitization by hyperthermia can be obtained irrespective of the Ku80 or DNA-PKcs status in cells. Therefore, Ku80 or DNA-PKcs and hence nonhomologous DSB end-joining do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.

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