Genomic integrity is continuously threatened by endogenous and exogenous agents that damage DNA and have immediate and long-term effects on cellular functioning. Replication of damaged DNA can cause mutations that may ultimately lead to carcinogenic events or, when occurring in germ cells, to inborn defects. Immediate effects of DNA damage include a blockage to transcription which can result in cell death by apoptosis (22). To minimize the harmful effects of DNA damage, nature has equipped cells with a sophisticated network of DNA repair mechanisms. Nucleotide excision repair (NER) is a cut-and-paste repair mechanism for the removal of a variety of bulky DNA lesions, such as chemical adducts and UV-induced photolesions (reviewed in references 11 and 16). The NER apparatus utilizes approximately 30 gene products to (i) recognize DNA damage, (ii) introduce single-stranded nicks 5′ and 3′ to the lesion, resulting in the removal of a single-stranded oligonucleotide containing the lesion, and (iii) restore the integrity of the double helix by gap-filling DNA synthesis (using the undamaged DNA strand as a template) and strand ligation (16). NER can be divided into two subpathways that differ in the molecular mechanism of lesion recognition. The global genome repair machinery (ggNER), in which damage recognition is performed by the XPC-HR23B complex (54), can remove lesions located anywhere in the genome. However, some types of lesions are poorly recognized by the ggNER apparatus and therefore are inefficiently repaired. Such lesions, when present in the template strand of active genes, interfere with transcription and activate the transcription-coupled repair subpathway of NER (tcNER). In the tcNER reaction, stalling of RNA polymerase II at bulky lesions serves as the damage sensor and subsequently recruits the remainder of the NER apparatus (37, 38, 55, 56). The importance of NER for genome preservation is illustrated by rare autosomal recessive disorders like xeroderma pigmentosum (XP), which is made up of seven complementation groups (XPA through XPG), and Cockayne syndrome (CS), which is made up of two complementation groups (CSA and CSB) (5). Both disorders are characterized by hypersensitivity to solar (UV) light. XP individuals demonstrate an increased rate of sunlight-induced skin cancer as well as carcinogen-induced internal tumors (5, 34). Except for XPC and XPE, where only the ggNER pathway is affected, XP is associated with a defect in both ggNER and tcNER. Individuals with CS exhibit small stature, intellectual impairment, cachexia, and sun sensitivity but no increased incidence of cancer (5, 15, 24). Cells from CS patients have been shown to be deficient in tcNER only (11, 16, 26, 57). An important question is why CS patients, despite their DNA repair deficiency, have not been reported to develop cancer of the skin or internal organs. A possible explanation is that CS cells can still perform ggNER, which, even though some types of lesions may be repaired at lower rates, could prevent accumulation of mutagenic lesions (15). Alternatively, since CSA and CSB cells cannot appropriately clear RNA polymerase II arrested at intragenic DNA lesions (39) and since stalled RNA polymerase II has been shown to promote intracellular accumulation of p53 and apoptosis (41, 53, 62), it has been suggested that in CS patients precancerous cells are more efficiently eliminated by apoptosis than they are in healthy persons (24). Among the growing number of mouse mutants with engineered deletions of genes involved in cellular responses to DNA damage are mouse models for XPA (18, 42), CSA (21), and CSB (59). Totally NER-deficient XPA−/− mice resemble human XPA patients in their UV-induced skin cancer predisposition (18, 42) and also show elevated frequencies of skin and internal tumors after exposure to chemical carcinogens (17, 18). In contrast to human CS, CSB−/− mice demonstrate an elevated incidence of both UV- and chemically induced skin cancer (59). This difference between CSB patients and mice might be due to the lack of expression of the p53-inducible p48 gene, required for ggNER of UV-induced cyclobutane dimers (and presumably other carcinogen-induced DNA lesions) (27). Since removal of these types of lesions in rodents almost totally depends on tcNER, the CSB defect may have a more dramatic effect in mice than in humans. Alternatively, since UV-induced skin tumor formation in CSB mice requires a longer latency time than that required by totally NER-deficient XPA mice (3, 4), CSB patients may simply not get old enough (average life span, 12.5 years) to develop tumors (43). Totally NER-deficient XPA mice, like human XP patients, develop spontaneous internal tumors upon aging (17). With the observation that, at least in mice, a specific tcNER defect predisposes to UV- and chemically induced skin cancer, the question of whether CS is also associated with increased sensitivity to spontaneous tumor formation arises. Mice with a homozygous disruption of the Ink4a/ARF locus have proven to be a particularly useful model system for studying factors (such as Ras and telomerase) contributing to neoplasia (9, 23, 48, 49). A great deal of evidence indicates that deletion of this region predisposes to cancer development (reviewed in references 8, 50, and 51). Humans with lesions in this genetic locus have an elevated incidence of melanoma, pancreatic carcinoma, and other neoplasms. Mice with homozygous disruption of the Ink4a/ARF locus are predisposed to developing spontaneous lymphomas and fibrosarcomas (49). The Ink4a gene product, p16Ink4a, helps regulate cellular proliferation by inhibiting cyclin-dependent kinases 4 and 6. The ARF gene product, p19ARF, is encoded by a gene overlapping that of the Ink4a gene but utilizing an alternative reading frame (ARF). p19ARF potentiates the function of p53 by antagonizing mdm2 function (52). If left unimpeded, mdm2 inhibits p53 function both by binding and by blocking p53's transcriptional activation domain as well as by catalyzing p53's ubiquitination, thus leading to its proteasomal degradation (32). Cancer-predisposed Ink4a/ARF−/− mice could therefore be used to investigate the role of the CSB gene product in spontaneous tumorigenesis. In the present study we have generated CSB−/− Ink4a/ARF−/− mice to study the effect of a CSB deficiency on spontaneous tumor formation. Surprisingly, since DNA repair genes are usually considered tumor suppressor genes, we found that inactivation of the CSB gene reduced spontaneous tumor formation in Ink4a/ARF−/− mice. Comparison of CSB−/− Ink4a/ARF−/−, CSB−/−, Ink4a/ARF−/−, and wild-type (WT) mouse embryo fibroblasts (MEFs) for their colony formation rates after low density seeding, H-Ras oncogene-mediated transformation rates, proliferation rates, and mRNA transcription rates revealed that at the cellular level, the CSB gene deficiency diminished neoplastic potential. Moreover, we show that the CSB gene deficiency sensitizes Ink4a/ARF−/− MEFs to UV-induced accumulation of p53 and apoptosis. Similarly, the CSB defect is shown to reduce the neoplastic potential of p53−/− MEFs. The implications of a CSB deficiency for tumorigenesis are discussed.