DNA-protein cross-links: Formidable challenges to maintaining genome integrity.

DNA is associated with proteins that are involved in its folding and transaction processes. When cells are exposed to chemical cross-linking agents or free radical-generating ionizing radiation, DNA-associated proteins are covalently trapped within the DNA to produce DNA-protein cross-links (DPCs). DPCs produced by these agents contain cross-linked proteins in an undisrupted DNA strand. Some DNA-metabolizing enzymes that form covalent reaction intermediates can also be irreversibly trapped in the presence of inhibitors or DNA damage to give rise to abortive DPCs. The abortive DPCs often contain cross-linked proteins attached to the 5' or 3' end of a DNA strand break. In vitro studies show that steric hindrance caused by cross-linked proteins impedes the progression of DNA helicases and polymerases and of RNA polymerases. The modes and consequences by which DPCs impede replication and transcription processes are considerably different from those with conventional DNA lesions. Thus, DPCs are formidable challenges to maintaining genome integrity and faithful gene expression. Current models of DPC repair involve direct and indirect removal of DPCs. The direct mechanism works for DPCs that contain topoisomerase 2 attached to the 5' end of DNA. The Mre11-Rad50-Nbs1 complex cleaves the site internal to the DPC and directly releases a DPC-containing oligonucleotide. The indirect mechanism involves degradation of cross-linked proteins by proteasomes or the recently identified DPC proteases Wss1 and Sprtn to relieve steric hindrance of DPCs. The resulting peptide-cross-links might be processed by translesion synthesis or other canonical repair mechanisms: however, the exact mechanism remains to be elucidated.

Radiation-induced DNA-protein cross-links: Mechanisms and biological significance.

Ionizing radiation produces various DNA lesions such as base damage, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, the biological significance of DPCs remains elusive. In this article, we focus on radiation-induced DPCs and review the current understanding of their induction, properties, repair, and biological consequences. When cells are irradiated, the formation of base damage, SSBs, and DSBs are promoted in the presence of oxygen. Conversely, that of DPCs is promoted in the absence of oxygen, suggesting their importance in hypoxic cells, such as those present in tumors. DNA and protein radicals generated by hydroxyl radicals (i.e., indirect effect) are responsible for DPC formation. In addition, DPCs can also be formed from guanine radical cations generated by the direct effect. Actin, histones, and other proteins have been identified as cross-linked proteins. Also, covalent linkages between DNA and protein constituents such as thymine-lysine and guanine-lysine have been identified and their structures are proposed. In irradiated cells and tissues, DPCs are repaired in a biphasic manner, consisting of fast and slow components. The half-time for the fast component is 20min-2h and that for the slow component is 2-70h. Notably, radiation-induced DPCs are repaired more slowly than DSBs. Homologous recombination plays a pivotal role in the repair of radiation-induced DPCs as well as DSBs. Recently, a novel mechanism of DPC repair mediated by a DPC protease was reported, wherein the resulting DNA-peptide cross-links were bypassed by translesion synthesis. The replication and transcription of DPC-bearing reporter plasmids are inhibited in cells, suggesting that DPCs are potentially lethal lesions. However, whether DPCs are mutagenic and induce gross chromosomal alterations remains to be determined.

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,ß-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,ß-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage.

Induction of DNA-protein cross-links by ionizing radiation and their elimination from the genome.

Ionizing radiation produces various types of DNA lesions, such as base damage, single-strand breaks, double-strand breaks (DSBs), and DNA-protein cross-links (DPCs). Of these, DSBs are the most critical lesions underlying the lethal effects of ionizing radiation. With DPCs, proteins covalently trapped in DNA constitute strong roadblocks to replication and transcription machineries, and hence can be lethal to cells. The formation of DPCs by ionizing radiation is promoted in the absence of oxygen, whereas that of DSBs is retarded. Accordingly, the contribution of DPCs to the lethal events in irradiated cells may not be negligible for hypoxic cells, such as those present in tumors. However, the role of DPCs in the lethal effects of ionizing radiation remains largely equivocal. In the present study, normoxic and hypoxic mouse tumors were irradiated with X-rays [low linear energy transfer (LET) radiation] and carbon (C)-ion beams (high LET radiation), and the resulting induction of DPCs and DSBs and their removal from the genome were analyzed. X-rays and C-ion beams produced more DPCs in hypoxic tumors than in normoxic tumors. Interestingly, the yield of DPCs was slightly but statistically significantly greater (1.3- to 1.5-fold) for C-ion beams than for X-rays. Both X-rays and C-ion beams generated two types of DPC that differed according to their rate of removal from the genome. This was also the case for DSBs. The half-lives of the rapidly removed components of DPCs and DSBs were similar (<1 h), but those of the slowly removed components of DPCs and DSBs were markedly different (3.9-5 h for DSBs versus 63-70 h for DPCs). The long half-life and abundance of the slowly removed DPCs render them persistent in DNA, which may impede DNA transactions and confer deleterious effects on cells in conjunction with DSBs.

Translocation and stability of replicative DNA helicases upon encountering DNA-protein cross-links.

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.

Repair and biochemical effects of DNA-protein crosslinks.

Genomic DNA is associated with various structural, regulatory, and transaction proteins. The dynamic and reversible association between proteins and DNA ensures the accurate expression and propagation of genetic information. However, various endogenous, environmental, and chemotherapeutic agents induce DNA-protein crosslinks (DPCs), and hence covalently trap proteins on DNA. Since DPCs are extremely large compared to conventional DNA lesions, they probably impair many aspects of DNA transactions such as replication, transcription, and repair due to steric hindrance. Recent genetic and biochemical studies have shed light on the elaborate molecular mechanism by which cells repair or tolerate DPCs. This review summarizes the current knowledge regarding the repair and biochemical effects of the most ubiquitous form of DPCs, which are associated with no flanked DNA strand breaks. In bacteria small DPCs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed by RecBCD-dependent homologous recombination (HR). NER does not participate in the repair of DPCs in mammalian cells, since the upper size limit of DPCs amenable to mammalian NER is smaller than that of bacterial NER. Thus, DPCs are processed exclusively by HR. The reactivation of the stalled replication fork at DPCs by HR seems to involve fork breakage in mammalian cells but not in bacterial cells. In addition, recent proteomic studies have identified the numbers of proteins in DPCs induced by environmental and chemotherapeutic agents. However, it remains largely elusive how DPCs affect replication and transcription at the molecular level. Considering the extremely large nature of DPCs, it is possible that they impede the progression of replication and transcription machineries by mechanisms different from those for conventional DNA lesions. This might also be true for the DNA damage response and signaling mechanism.

Comparison of the activities of bacterial and mammalian nucleotide excision repair systems for DNA-protein crosslinks.

Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.

Genetic analysis of repair and damage tolerance mechanisms for DNA-protein cross-links in Escherichia coli.

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in Escherichia coli. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome.

Repair of DNA-protein crosslink damage: coordinated actions of nucleotide excision repair and homologous recombination.

DNA-protein crosslinks (DPCs) are extremely bulky DNA lesions, and steric hindrance imposed by covalently trapped proteins would hamper the transaction of DNA such as replication, transcription, and repair. However, it has been largely elusive how cells mitigate the genotoxic effect of DPCs. We have recently shown that nucleotide excision repair (NER) and homologous recombination (HR) differentially contribute to the repair of DPCs in E. coli cells. Several lines of genetic and biochemical evidence indicate that NER repairs DPCs with crosslinked proteins (CLPs) of sizes less than 12-14 kDa, whereas DPCs with oversized CLPs are processed exclusively by RecBCD-dependent HR. The present result shows that cells use the coordinated actions of NER and HR to deal with unusually bulky DNA lesions like DPCs.

Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.

DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.