Formation and repair of DNA-protein crosslinks in newly replicated DNA

The production and removal of gamma-radiation-induced DNA-protein crosslinks (DPC) in nuclear matrix-associated newly replicated DNA were examined, as well as the relationship of DPC to DNA replication. In unirradiated, exponentially growing Chinese hamster V79 cells, DNA pulse labeled with [3H]thymidine was observed to be bound preferentially to protein. The pulse-labeled DNA subsequently became dissociated from protein. After a 30- to 60-min chase period, the level of labeled DNA in DPC was reduced to the same level as for bulk DNA. The radiation dose response for the formation of DPC was similar in newly replicated DNA that had been chased for various times and in mature chromatin DNA. Labeled DNA, in the DPC formed after 60 Gy, was rapidly removed from protein during the postirradiation incubation period. However, no recovery of DNA synthesis was observed, even after the majority of DPC were released. Thus either DPC are not the sole cause of the inhibition of DNA synthesis or their removal is not sufficient for DNA synthesis to resume.     

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

DNA-protein crosslinks: their induction, repair, and biological consequences

The covalent crosslinking of proteins to DNA presents a major physical challenge to the DNA metabolic machinery. DNA-protein crosslinks (DPCs) are induced by a variety of endogenous and exogenous agents (including, paradoxically, agents that are known to cause cancer as well as agents that are used to treat cancer), and yet they have not received as much attention as other types of DNA damage. This review summarizes the current state of knowledge of DPCs in terms of their induction, structures, biological consequences and possible mechanisms of repair. DPCs can be formed through several different chemistries, which is likely to affect the stability and repair of these lesions, as well as their biological consequences. The considerable discrepancy in the DPC literature reflects both the varying chemistries of this heterogeneous group of lesions and the fact that a number of different methods have been used for their analysis. In particular, research in this area has long been hampered by the inability to chemically define these lesions in intact cells and tissues. However, the emergence of proteomics as a tool for identifying specific proteins that become crosslinked to DNA has heralded a new era in our ability to study these lesions. Although there are still many unanswered questions, the identification of specific proteins crosslinked to DNA should facilitate our understanding of the down-stream effects of these lesions.     

An epigenetic code for DNA damage repair pathways?

Exposure of living cells to intracellular or external mutagens results in DNA damage. Accumulation of DNA damage can lead to serious consequences because of the deleterious mutation rate resulting in genomic instability, cellular senescence, and cell death. To counteract genotoxic stress, cells have developed several strategies to detect defects in DNA structure. The eukaryotic genomic DNA is packaged through histone and nonhistone proteins into a highly condensed structure termed chromatin. Therefore the cellular enzymatic machineries responsible for DNA replication, recombination, and repair must circumvent this natural barrier in order to gain access to the DNA. Several studies have demonstrated that histone/chromatin modifications such as acetylation, methylation, and phosphorylation play crucial roles in DNA repair processes. This review will summarize the recent data that suggest a regulatory role of the epigenetic code in DNA repair processes. We will mainly focus on different covalent reversible modifications of histones as an initial step in early response to DNA damage and subsequent DNA repair. Special focus on a potential epigenetic histone code for these processes will be given in the last section. We also discuss new technologies and strategies to elucidate the putative epigenetic code for each of the DNA repair processes discussed.     

Brc1-mediated DNA repair and damage tolerance

The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1Delta cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1Delta epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination.     

DNA damage tolerance,mismatch repair and genome instability

DNA mismatch repair is an important pathway of mutation avoidance. It also contributes to the cytotoxic effects of some kinds of DNA damage, and cells defective in mismatch repair are resistant, or tolerant, to the presence of some normally cytotoxic base analogues in their DNA. The absence of a particular mismatch binding function from some mammalian cells confers resistance to the base analogues O⁶-methylguanine and 6-thioguanine in DNA. Cells also acquire a spontaneous mutator phenotype as a consequence of this defect. Impaired mismatch binding can cause an instability in DNA microsatellite regions that comprise repeated dinucleotides. Microsatellite DNA instability is common in familial and sporadic colon carcinomas as well as in a number of other tumours. Several independent lines of investigation have identified defects in mismatch repair proteins that are causally related to these cancers.     

DNA interstrand crosslinks repair in mammalian cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.     

Radiation damage to DNA in DNA-protein complexes

The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals.     

Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli.

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.     

Identification of human genes involved in repair and tolerance of DNA damage

Summary An overview is presented on strategies of cloning mammalian DNA repair genes. Complementation of human and rodent repair defects and mutagen hypersensitivities by chromosome and DNA mediated gene transfer and mRNA microinjection is described, and the features of the cloned human DNA repair genes are summarized. It is shown that transfection of repair deficient cell lines with cloned bacterial and human genes may give rise to protection from the genotoxic effects of mutagens.Abbreviations MGMT O⁶-methylguanine-DNA methyltransferase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HeCNU N-hydroxyethyl-N-chloroethylnitrosourea - MMC mitomycin C - MPA mycophenolic acid - ERCC excision-repair cross-complementing rodent UV-complementation group - UV ultraviolet light - XP xeroderma pigmentosum - CHO Chinese hamster ovary Dedicated to Prof. Dr. U. Hagen on the occasion of his 65th birthdayExtended version of an oral presentation given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Cellular senescence and DNA repair

Aging is a complex process that results in functional decline and mortality of the organisms. On the cellular lever, cellular senescence has been used as a model for aging. Therefore, understanding cellular senescence has important health implications. Initial observations suggest that cellular senescence is the result of telomere shortening. Recent findings suggest that cellular senescence could be triggered by DNA damage. In fact, both telomere shortening and DNA-damage-induced cellular senescence share a common mechanism, the DNA damage response pathway. This review will discuss the link between DNA repair defects and cellular senescence.     

Enhanced repair of DNA interstrand crosslinks in S phase

Hela cells synchronized in G1 and S phases of the cell cycle were transfected with pEGFP crosslinked with trioxsalen. Twelve hours later the number of fluorescent cells was determined by fluorescent microscopy. Cells in S phase have repaired 0.2-0.3 ICL/kb over the 12h period, while cells in G1 phase repaired interstrand crosslinks much more poorly. The crosslinked plasmids were efficiently recruited to the nuclear matrix both in G1 phase and S-phase, which showed that the poor repair of G1 cells was a result of a lack of DNA replication rather than of a lack of matrix attachment.     

甲醛对DNA损伤的彗星实验研究

甲醛是一种遗传毒性物质。国内外学者的大量研究证实,甲醛可以引起DNA-DNA、DNA-蛋白质分子交联,但对于甲醛是否能够引起DNA分子的断裂,学界却存在分歧。本实验以颊黏膜细胞作为实验材料,通过彗星实验对甲醛的遗传毒性——尤其是DNA分子断裂作用进行了系统的研究。结果显示甲醛在较低浓度(5μmol／L,7,5μmol／L,10μmol／L)时具有断裂作用,在较高浓度(15μmol／L,30μmol／L,50μmol／L)时则具有交联作用。根据本实验的结果,本文还首次论证了甲醛断裂作用的断裂峰值(7．5μmol／L)。