UV Light-induced DNA Damage and Tolerance for the Survival of Nucleotide Excision Repair-deficient Human Cells

Abstract

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase is involved in the tolerance of 6-4PPs in human cells.DNA lesions cause cell killing and mutations if they are not adequately repaired. Exposure of cells to UV light radiation results in formation of the two most common lesions, the cyclobutane pyrimidine dimer (CPD) 1 and the 6-4 pyrimidinepyrimidone photoproduct (6-4PP) at adjacent pyrimidines (1). These lesions inhibit DNA replication and transcription and are normally removed from the genome by nucleotide excision repair (NER) in most organisms, including humans (2). By contrast to a cis-syn CPD, which has only a modest effect on the DNA structure, a 6-4PP induces a large structural distortion and is repaired more rapidly than a CPD by NER (3).It is becoming widely appreciated that cells have developed a mechanism to tolerate damage in their genome without removing it. NER-deficient cells, such as XP12ROSV belonging to the xeroderma pigmentosum A (XP-A) group, for example, cannot remove UV light-induced DNA damage from their genome at all; but after irradiation with a dose of 0.3 J/m 2 UV-C light, which creates Ͼ10 4 DNA lesions with a ratio of three CPDs to one 6-4PP in the genome, 50% of the XP-A cells continue to divide and form colonies (4). This tolerance mechanism is known as postreplication repair. In human cells, a major mechanism for carrying out postreplication repair is translesion synthesis (TLS), which is performed by specialized DNA polymerases at the site of DNA damage (5). The gene defective in XP variants (XP-V) encodes a DNA polymerase (pol ), which can replicate undamaged and CPD-containing templates with equal efficiency in vitro (6, 7). Although several DNA polymerases involved in TLS have been identified in vitro, it is still unclear which polymerase is responsible for the bypass of various types of DNA damage, including 6-4PPs in vivo.Many DNA lesions encounter replica...

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

“…There results indicated that N. crassa possesses the excess activity for the removal of 6-4PPs. The ability to remove the 6-4PPs may be more important for tolerance to UV in N. crassa, because it is known that 6-4PPs are more toxic than CPDs in NER-deficient human cells (Nakajima et al, 2004). Moreover N. crassa has a CPD specific photolyase which functions under the light condition, suggesting that N. crassa also possesses the excess activity for the repair of CPDs (Shimura et al, 1999).…”

Differential effects of NF-κB on apoptosis induced by DNA-damaging agents: the type of DNA damage determines the final outcome

“…One of the unique features of UVB radiation is its capacity to induce DNA damage, preferentially CPDs and /6-4S photoproducts (Nakajima et al, 2004). It is known that UVB-induced DNA damage plays an important role in the signal transduction of a variety of biological effects of UVB radiation, as reduction of DNA damage, for example, via the application of exogenous DNA repair enzymes, is frequently associated with a loss or reduction of the respective biological effect .…”

“…UVB-induced apoptosis is regarded as a protective mechanism preventing the generation of skin cancer by eliminating cells that harbour high amounts of UVB-induced DNA damage. Accordingly, UVB-induced DNA damage (cyclobutane pyrimidine dimers, o6-4> photoproducts) has been demonstrated to be the major molecular trigger for UVB-mediated cell death (Nakajima et al, 2004). However, it has also been recognized that activation of death receptors (CD95/ Fas; tumour necrosis factor receptor 1 (TNF-R1); tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-R) and generation of reactive oxygen species contribute to the complete apoptotic response in an independent but additive fashion .…”

Resposta a danos no DNA após exposição à luz ultravioleta: apagando o fogo antes do incêndio celular

“…comparado às polimerases clássicas da família B (McCulloch e Kunkel, 2008), entretanto, dependendo da polimerase TLS recrutada, a lesão pode ser replicada praticamente sem erro como Pol η tendo CPD de timinas (T^T) como molde (Johnson et al, 2000). A outra lesão gerada por luz UV, o díme-ro 6-4PP, possui uma distorção muito maior e não pode ser replicada por Pol η e estudos indicam que Pol ζ e REV1 são necessárias para transpor essa lesão, porém com maior taxa de mutagênese (Nakajima et al, 2004).…”