Additional Repair Mechanisms Research Articles

Annexins A1 and A2 are recruited to larger lysosomal injuries independently of ESCRTs to promote repair.

Damaged lysosomes can be repaired by calcium release-dependent recruitment of the ESCRT machinery. However, the involvement of annexins, another group of calcium-responding membrane repair proteins, has not been fully addressed. Here, we show that although all ubiquitously expressed annexins (ANXA1, A2, A4, A5, A6, A7, and A11) localize to damaged lysosomes, only ANXA1 and ANXA2 are important for repair. Their recruitment is calcium-dependent, ESCRT-independent, and selective towards lysosomes with large injuries. Lysosomal leakage was more severe when ANXA1 or ANXA2 was depleted compared to that of ESCRT components. These findings suggest that ANXA1 and ANXA2 constitute an additional repair mechanism that serves to minimize leakage from damaged lysosomes.

Combined Proteomic and Physiological Analysis of Chloroplasts Reveals Drought and Recovery Response Mechanisms in Nicotiana benthamiana.

Chloroplasts play essential roles in plant metabolic processes and stress responses by functioning as environmental sensors. Understanding chloroplast responses to drought stress and subsequent recovery will help the ability to improve stress tolerance in plants. Here, a combined proteomic and physiological approach was used to investigate the response mechanisms of Nicotiana benthamiana chloroplasts to drought stress and subsequent recovery. Early in the stress response, changes in stomatal movement were accompanied by immediate changes in protein synthesis to sustain the photosynthetic process. Thereafter, increasing drought stress seriously affected photosynthetic efficiency and led to altered expression of photosynthesis- and carbon-fixation-related proteins to protect the plants against photo-oxidative damage. Additional repair mechanisms were activated at the early stage of recovery to restore physiological functions and repair drought-induced damages, even while the negative effects of drought stress were still ongoing. Prolonging the re-watering period led to the gradual recovery of photosynthesis at both physiological and protein levels, indicating that a long repair process is required to restore plant function. Our findings provide a precise view of drought and recovery response mechanisms in N. benthamiana and serve as a reference for further investigation into the physiological and molecular mechanisms underlying plant drought tolerance.

Abstract 1361: Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer

Abstract The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of 22% of NSCLC to be FA functionally inactive by Fanconi Anemia Triple Staining Immunofluorescence (FATSI) test. Studies have shown involvement of certain micro RNA (miRNA) as regulatory elements in the development of lung cancer. We set out to evaluate potential involvement of miRNAs in the regulation of the Fanconi Anemia (FA) pathway. Using Nanostring counter miRNA array we screened 734 different miRNA expression in two FA defective lung cancer cells and matched control cells along with two FA pathway deficient lung tumors and matched non-tumor lung tissue samples. Selected miRNA expression were validated with real-time PCR analysis. miRNA target gene expression was analyzed through AmpliSeq RNA gene expression analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. Nanostring data showed that miR-200C was increased 7.5 fold on average in the FA defective lung cancer cells as compared to control cell. An average of 22 fold increase in miR-200C was detected in the FA defective lung tumor tissues comparing to matching non-tumor tissues. AmpliSeq analysis showed the ZEB1(zinc finger E-box binding homeobox 1) mRNA expression was down regulated in10 out 10 lung tumors (100%) comparing to non-tumor tissues, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibiting cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. Our findings indicate that the FA pathway regulates downstream genes through regulation of miRNAs in lung cancer. MiR-200C appears to be one of the most important FA downstream regulators in lung cancer. Validation with a larger sample size will be needed to confirm our findings. Citation Format: Wenrui Duan, Shirley Tang, Li Gao, Kathleen Dotts, Andrew Fink, Arjun Kalvala, Brittany Aguila, Miguel A. Villalona-Calero. Micro RNA-200C is one of the important Fanconi Anemia (FA) pathway downstream regulators in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1361.

Abstract 4442: micoRNA in FA defective tumor

Abstract The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in the endogenous repair of double stranded DNA breaks and homologous recombination as well as repair of DNA cross-linking caused by exogenous agents. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). MicroRNAs (miRs, miRNA) are small, 19-25 nucleotide, single stranded, non-coding RNA molecules that have regulatory roles in humans by targeting mRNAs via cleaved or translational repression which can affect the output of protein coding genes and cause various changes in developmental and physiological processes including cancer. It has been published that some miRNAs are tumor suppressors, and others are oncogenes. However, microRNAs and the FA pathway in relationship to lung cancer progression remain unknown. In order to elucidate the role of FA related miRs in human lung cancer development or treatment, we analyzed miR expression in lung cancer cells and patient lung tumors. Several cell lines were generated using RNAi technology with FANCD2 deficiency and confirmed via reduction in FANCD2 protein expression. Using Nanostring counter technology, microRNA array analysis was performed on FANCD2 foci positive and foci negative tumors as well as matching normal lung tissues. A cluster of microRNAs (mir-431, 377, 376c, 200c, 26a, 135a, and let-7e) were found to be up-regulated, and miR-494,205, 630, 145,671 were found to be down-regulated in the foci negative tumors samples compared to matching non-tumor lung tissue. The same group of miRs were upregulated and its corresponding group were down-regulated in the foci negative tumors as compared to foci positive tumors. These findings indicate that the FA pathway may regulate downstream genes through regulation of microRNAs in lung cancer. In order to validate this hypothesis, specific miRs were be analyzed with Real-Time Quantitative PCR via foci positive and foci negative samples (cell and patient tissue) in hopes of finding significant and consistent regulation. This study contributes to further understanding in miRNA and cancer pathology that may be applicable to futuristic lung cancer treatment. Citation Format: Shirley Tang, Li Gao, Kathleen Kathleen, Andrew Fink, Arjun Kalvala, Brittany Aguila, Gregory Otterson, Miguel Villalona-Calero, Wenrui Duan. micoRNA in FA defective tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4442. doi:10.1158/1538-7445.AM2017-4442

Abstract 4438: Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC)

Abstract Gene promoter methylation is an epigenetic mechanism used by cells to control gene expression. Over recent decades, scientists have made various discoveries linking DNA methylation to several adverse outcomes, including human cancers. The Fanconi Anemia (FA) pathway is involved in homologous recombination, one of the major mechanisms of DNA repair. This pathway is essential for human cells to maintain integrity following DNA damage. Cancers with defective FA pathways are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains at least 19 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. Promoter methylation in FA genes is thought to play a role in the occurrence of cancer. Recently we screened 139 non-small cell lung cancer (NSCLC) formalin-fixed, paraffin-embedded (FFPE) tumors for FANCD2 foci formation by FA triple stain immunofluorescence (FATSI) analysis. Among the 104 evaluable tumors, 23 (22%) were FANCD2 foci negative. Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated 39 NSCLC samples (21 foci positive and 18 foci negative; 21 adenocarcinomas, 17 squamous cell carcinomas, 1 large cell carcinoma) for FANCF, FANCL and FANCS (BRCA1) promoter methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University after IRB approval. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The promoter methylation status of FANCF, FANCL and FANCS was evaluated using methylation-specific PCR (MS-PCR). Among the 18 FATSI negative tumors, promoter methylation was found in FANCF (1 adenocarcinoma), FANCL (1 adenocarcinoma) and FANCS (1 adenocarcinoma). Among the 21 FATSI positive tumors, no promoter methylation was detected in FANCF or FANCL. Promoter methylation in FANCS was found in 1 (squamous cell carcinoma) of 21 FATSI positive tumors. The above observations suggest that epigenetic alterations, specifically methylation, can be one of the factors that contribute to FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Further studies in FA gene expression are needed to determine the impact of FA gene promoter methylation on FA repair foci formation. Citation Format: Andrew Fink, Arjun Kalvala, Li Gao, Kathleen Dotts, Brittany Aguila, Shirley Tang, Gregory A. Otterson, Miguel A. Villalona-Calero, Wenrui Duan. Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4438.

Fanconi anemia repair pathway dysfunction, a potential therapeutic target in lung cancer.

The Fanconi anemia (FA) pathway is a major mechanism of homologous recombination DNA repair. The functional readout of the pathway is activation through mono-ubiquitination of FANCD2 leading to nuclear foci of repair. We have recently developed an FA triple-staining immunofluorescence based method (FATSI) to evaluate FANCD2 foci formation in formalin fixed paraffin-embedded (FFPE) tumor samples. DNA-repair deficiencies have been considered of interest in lung cancer prevention, given the persistence of damage produced by cigarette smoke in this setting, as well as in treatment, given potential increased efficacy of DNA-damaging drugs. We screened 139 non-small cell lung cancer (NSCLC) FFPE tumors for FANCD2 foci formation by FATSI analysis. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative, thus repair deficient. To evaluate and compare novel-targeted agents in the background of FA deficiency, we utilized RNAi technology to render several lung cancer cell lines FANCD2 deficient. Successful FANCD2 knockdown was confirmed by reduction in the FANCD2 protein. Subsequently, we treated the FA defective H1299D2-down and A549D2-down NSCLC cells and their FA competent counterparts (empty vector controls) with the PARP inhibitors veliparib (ABT-888) (5 μM) and BMN673 (0.5 μM), as well as the CHK1 inhibitor Arry-575 at a dose of 0.5 μM. We also treated the FA defective small cell lung cancer cell lines H719D2-down and H792D2-down and their controls with the BCL-2/XL inhibitor ABT-263 at a dose of 2 μM. The treated cells were harvested at 24, 48, and 72 h post treatment. MTT cell viability analysis showed that each agent was more cytotoxic to the FANCD2 knock-down cells. In all tests, the FA defective lung cancer cells had less viable cells as comparing to controls 72 h post treatment. Both MTT and clonogenic analyses comparing the two PARP inhibitors, showed that BMN673 was more potent compared to veliparib. Given that FA pathway plays essential roles in response to DNA damage, our results suggest that a subset of lung cancer patients are likely to be more susceptible to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. These subjects can be identified through FATSI analysis. Clinical trials to evaluate this therapeutic concept are needed.

Stem cells for liver regeneration

The liver has a unique capacity to repair following injury, which is largely achieved by proliferation of hepatocytes. However, in situations of chronic or overwhelming liver injury, additional repair mechanisms, namely liver progenitor or oval cells, are activated. These cells, located in the canals of Hering, express markers for both hepatocytes and biliary cells and have the capacity to differentiate down both hepatocyte and biliary lineages. Previous work has suggested that the administration of autologous or allogeneic cell therapies such as haematopoietic or mesenchymal stem cells can augment liver repair by either stimulating endogenous repair mechanisms or by suppressing ongoing damage. A better understanding of how cell therapies can promote liver regeneration will lead to the refinement of these therapeutic approaches and also develop new pharmacological agents for liver repair.

Abstract 4246: Promoter hypermethylation and gene expression of FANCF in non-small cell lung cancer (NSCLC).

Abstract The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in repairing double stranded DNA breaks. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains 15 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. The methylation of CpG islands in the FANCF gene is thought to plays a role in the occurrence of ovarian cancer. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated a series of NSCLC samples for FANCF methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University Comprehensive Cancer Center after IRB approval. FA pathway status was evaluated by FATSI. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The methylation status of the FANCF gene promoter was evaluated using methylation-specific PCR (MS-PCR). FANCF gene expression was evaluated by NanoString assays. We screened total of 40 NSCL tumors by the FATSI assay, and the ratio of FANCD2 foci negative tumor was 20% (8/40). Squamous cell carcinoma, adenocarcinoma and large cell carcinoma histologies were all represented in the samples. FANCF promoter methylation was present in 2 of 8 FATSI negative tumors (1 adenocarcinoma and 1 large cell carcinoma) based on MS-PCR analysis, and absent in 8 tested FATSI positive tumors. NanoString analysis was performed in 5 FATSI positive and 5 FATSI negative tumors which the latter including the two tumors with FANCF methylation identified with the MS-PCR. FANCF mRNA level was 1.8-fold lower in tumors with promoter methylation as comparing to matched non-tumor tissues. One of the two tumors containing FANCF methylation was also analyzed with RNAseq, and the results showed a 1.6 fold reduction in FANCF mRNA in the tumor as comparing to matched non-tumor tissue. However the FANCF mRNA level was similar between tumor and non-tumor (tumor/non-tumor = 1.04) in the samples without methylation. The above observation suggests that epigenetic alterations can be the base for FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Evaluation of pre and post treatment samples in FATSI negative patients undergoing therapy would be necessary to support this hypothesis. Citation Format: Wenrui Duan, Tyler Rees, Kevin Vu, Brittany Barnwell, Li Gao, Arjun Kalvala, xin wu, Gregory A. Otterson, Miguel A. Villalona-Calero. Promoter hypermethylation and gene expression of FANCF in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4246. doi:10.1158/1538-7445.AM2013-4246

Abstract 5500: Inhibition of poly(ADP-ribose) polymerase in lung cancer cells with defective Fanconi anemia (FA) pathway

Abstract The poly ADP-ribose polymerase (PARP) inhibitors are novel agents being developed with the hope of inhibiting base excision repair, a process that is of prime importance for tumors to survive genotoxic insult. BRCA gene homozygous deficiency has been identified as a potential predictor of response to PARP inhibitors. BRCA is involved in homologous recombination (HR), an example of double-strand break repair. Thus, inhibiting tumors which have lost one DNA repair pathway by targeting a second DNA repair pathway represents groundbreaking therapeutic strategy. BRCA is just one of many genes that collaborate in the same repair pathway, which has been named the Fanconi Anemia (FA) pathway. The FA pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Deficiencies in FA pathway have been reported as a predictor of cisplatin and mitomycin C (MMC) sensitivity in cancer cells. Given that this pathway plays essential roles in response to the DNA damage, cancers with defective FA pathway are expected to be more sensitive to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. In order to preclinically evaluate the effect of PARP inhibition in lung cancer cells with spontaneously defective FA pathway, we utilized RNAi technology to create FANCD2 knockdown cancer cells. Lung cancer cells H1299 and A549 were transduced with FANCD2-specific shRNA-expressing and puromycin-resistant lentiviral particles, or control shRNA lentiviral particles to create stably transduced cells. Successful FANCD2 knockdown was confirmed by the Western blot by reduction in the FANCD2 protein. Cell viability was evaluated with MTT (Dimethylthiazolyl-2-5-diphenyltetrazolium bromide) analysis. The FA defective H1299 and A549 lung cancer cells and the controls (transected with empty vectors) were treated with the PARP inhibitor ABT888 at a dose of 5µM. Then the treated cells were harvested at 24, 48 and 72 hours post treatment. MTT cell viability analysis showed that ABT888 alone was cytotoxic to the FA deficient lung cancer cells (H1299D2-down and A549D2-down) 72 hours post treatment. In both cell types, the FA defective lung cancer cells had less viable cells compared to controls 72 hours post treatment. Recent clinical data confirmed the PARP inhibitors could be used not only as chemosensitizers but as well as single agents to selective kill tumors with BRCA-deficient breast cancers. Disruption of FA cascade has been reported in solid tumors. Given that the FA pathway plays an essential role in response to therapy-induced DNA interstrand cross-links, it is very plausible that cancers with defective FA pathway are more sensitive to cross-link based therapy and that treatments like PARP inhibitors in which an additional repair mechanism is targeted. A clinical trial to validate this hypothesis is under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2011-5500

Abstract 3750: Functional analysis of the Fanconi Anemia pathway in primary lung tumors

Abstract The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Lack of formation of FANCD2 foci has been reported as a predictor of cisplatin and mitomycin C (MMC) sensitivity in cancer cells. Given that this pathway plays essential roles in response to the DNA damage, cancers with defective FA pathway are expected to be more sensitive to DNA cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. We have developed a test to identify tumors with defective FA pathway using paraffin embedded tissues. Paraffin embedded lung primary cancer specimens were subjected to immunofluorescence microscopy (IF) analysis to evaluate the status of FancD2 subnuclear foci formation, Ki67 protein expression and DAPI staining of nuclei simultaneously in order to eliminate false negative tumor specimens due to absence of tumor cells or low proliferation. Additional validation using FA defective PD220 and PD20 cell-lines as well as fresh tumor tissues was done; IF analysis and Western blotting analysis of FDACD2 protein mono-ubiqutination were performed. The ratio of FANCD2 foci negative tumor was 20% among 20 paraffin embedded non-small cell lung cancer samples analyzed. Histological characteristics of the defective tumors include large cell carcinoma and adenocarcinoma. The proposed immunofluorecence staining method evaluates functional status of FA pathway using paraffin embedded tissues even after long term storage. Given the clinical availability of agents targeting alternative mechanisms of repair, the status of FA pathway may be an important biomarker for personalized therapeutic treatment in patients with lung cancers. A clinical trial to validate this hypothesis is under way. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3750.

Mucosal repair and growth factors: recombinant human hepatocyte growth factor as an innovative therapy for inflammatory bowel disease

The repair of intestinal mucosal injuries is a tightly regulated process involving epithelial restitution, cell proliferation and maturation, and the dedifferentiation of epithelial cells. Deeper injuries also require additional repair mechanisms, including inflammatory processes, angiogenesis, and extracellular-matrix deposition. Once intestinal mucosal injury occurs, numerous growth factors and cytokines, including hepatocyte growth factor (HGF), keratinocyte growth factor, endothelial growth factor, epidermal growth factor, transforming growth factor-beta1, intestinal trefoil factor, interleukin (IL)-1, and IL-2, are induced in both the intestinal lumen and submucosa, and these factors cooperatively stimulate epithelial mucosal repair. HGF, a major agent promoting hepatocyte proliferation, also modulates intestinal epithelial cell proliferation and migration, leading to the acceleration of intestinal mucosal repair. Additionally, the proteolytic activation of HGF, which is mediated by HGF activator, is essential for the regeneration of injured intestinal mucosa. Recently, several studies have shown that the administration of recombinant human HGF or HGF gene therapy abrogates disease severity in several animal models of inflammatory bowel disease (IBD). Recombinant human HGF will soon be available for administration to patients with fulminant hepatic failure. Although additional preclinical biological studies are required, HGF has the potential to be an important new treatment modality promoting intestinal mucosal repair in patients with IBD.

Formation and persistence of N7- and O6-methyl-guanine in DNA of chick embryo brain cells in ovo following administration of N-nitroso-N-methylurea.

Previously, O6-methyl-guanine-DNA alkyltransferase (AT) of the chicken embryo has been investigated in vitro. In the present studies, the effect of in vivo (in ovo) treatment with methylnitrosourea (MNU) was examined at a developmental stage of 15 days and doses of 1.25-20 mg/egg, yielding about 1-16 mmol MNU/kg embryo weight. At intervals of 1-24 h, DNA of the brain was prepared. N7-methylguanine and O6-methylguanine were quantified by combining a rapid method of DNA isolation, high-pressure-liquid-chromatography (HPLC) and electrochemical detection of the guanine-alkyl adducts. In parallel, the AT activity of brain homogenates was determined. Within the range of the detection limits (N7-methylguanine: 16 nM, O6-methylguanine: 2.5 nM), no repair of the guanine adducts, being about 500 nmol O6-methyl- and 1800 nmol N7-methyl-adducts per mmol guanine 1 h following administration of 10 mg MNU/egg, was evident. The rather low acute toxicity of MNU in the chicken embryo at the 15th day of development DL50/24 h being > 4 mg MNU/embryo, argues, therefore, for an additional repair mechanism, e.g. cell replacement repair.

Differential repair of UV damage in Saccharomyces cerevisiae is cell cycle dependent.

In the yeast Saccharomyces cerevisiae the transcriptionally active MAT alpha locus is repaired preferentially to the inactive HML alpha locus after UV irradiation. Here we analysed the repair of both loci after irradiating yeast cells at different stages of the mitotic cell cycle. In all stages repair of the active MAT alpha locus occurs at a rate of 30% removal of dimers per hour after a UV dose of 60 J/m2. The inactive HML alpha is repaired as efficiently as MAT alpha following irradiation in G2 whereas repair of HML alpha is less efficient in the other stages. Thus differential repair is observed in G1 and S but not in G2. Apparently, in G2 a chromatin structure exists in which repair does not discriminate between transcriptionally active and inactive DNA or, alternatively, an additional repair mechanism might exist which is only operational during G2.