ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines

In mammalian nucleotide excision repair (NER), the ERCC1 protein is known to act as a complex with ERCC4 (XPF) protein, which is necessary for stability of ERCC1, and this complex introduces an incision on the 5′ side of a damaged site in DNA. ERCC1 also binds to XPA protein to make a large protein complex at the site of DNA damage. Since no human disease associated with ERCC1 has been identified, Chinese hamster ovary (CHO) cell lines defective in ERCC1 are a unique source for characterization of ERCC1 deficiency in mammalian cells. We have isolated the full length ERCC1 cDNA from a wild-type CHO cell line and analyzed mutations in two CHO cell lines which fall into complementation group 1 of UV-sensitive rodent cell lines. One cell line, 43-3B, has a missense mutation at the 98th residue (V98E). The in vitro translated mutant protein of 43-3B is unable to bind to XPA protein. Although the mutant protein is able to bind to XPF protein in vitro, the mutant protein is highly unstable in vivo. These defects presumably cause the NER deficiency of this cell line. Another mutant, UV-4, has an insertion mutation in the middle of the coding sequence, resulting in a truncated protein due to a nonsense codon arising from the frameshift. Thus, these two mutant cell lines are deficient in the function of the ERCC1 gene for NER.     

Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines

We have previously shown that the alkaline Comet assay (single cell gel electrophoresis) in a modified version is a sensitive test for the detection of formaldehyde-induced DNA-protein crosslinks (DPC). Our results also indicated that formaldehyde-induced DPC are related to the formation of chromosomal effects such as micronuclei and sister chromatid exchanges. To better understand the genetic consequences of formaldehyde-induced DPC we have now investigated the induction and removal of DPC in relationship to the formation of micronuclei in normal and repair-deficient human cell lines. We did not find significant differences between normal cells, a xeroderma pigmentosum (XP) cell line and a Fanconi anaemia (FA) cell line with respect to the induction and removal of DPC. However, the induction of micronuclei was enhanced in both repair-deficient cell lines, particularly in XP cells, under the same treatment conditions. Comparative investigations with the DNA-DNA crosslinker mitomycin C (MMC) revealed a delayed removal of crosslinks and enhanced induction of micronuclei in both repair-deficient cell lines. FA cells were found to be particularly hypersensitive to micronucleus induction by MMC. In contrast to the results with formaldehyde, induction of micronuclei by MMC occurred at much lower concentrations than the effects in the Comet assay. Our results suggest that more than one repair pathway can be involved in the repair of crosslinks and that disturbed excision repair has more severe consequences with regard to the formation of chromosomal aberrations after formaldehyde treatment than has disturbed crosslink repair.     

ERCC1 is required for FANCD2 focus formation

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.     

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.     

Mammalian SNM1 is required for genome stability

The protein encoded by SNM1 in Saccharomyces cerevisiae has been shown to act specifically in DNA interstrand crosslinks (ICL) repair. There are five mammalian homologs of SNM1, including Artemis, which is involved in V(D)J recombination. Cells from mice constructed with a disruption in the Snm1 gene are sensitive to the DNA interstrand crosslinker, mitomycin (MMC), as indicated by increased radial formation following exposure. The mice reproduce normally and have normal life spans. However, a partial perinatal lethality, not seen in either homozygous mutant alone, can be noted when the Snm1 disruption is combined with a Fancd2 disruption. To explore the role of hSNM1 and its homologs in ICL repair in human cells, we used siRNA depletion in human fibroblasts, with cell survival and chromosome radials as the end points for sensitivity following treatment with MMC. Depletion of hSNM1 increases sensitivity to ICLs as detected by both end points, while depletion of Artemis does not. Thus hSNM1 is active in maintenance of genome stability following ICL formation. To evaluate the epistatic relationship between hSNM1 and other ICL repair pathways, we depleted hSNM1 in Fanconi anemia (FA) cells, which are inherently sensitive to ICLs. Depletion of hSNM1 in an FA cell line produces additive sensitivity for MMC. Further, mono-ubiquitination of FANCD2, an endpoint of the FA pathway, is not disturbed by depletion of hSNM1 in normal cells. Thus, hSNM1 appears to represent a second pathway for genome stability, distinct from the FA pathway.     

Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells

The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.     

DNA sequence analysis of mutations induced by melphalan in the CHO aprt locus.

In previous work, we established that treatment with melphalan (L-phenylalanine mustard) produced a predominance of A.T-->T.A transversions in the Simian virus 40 (SV40)-based shuttle vector pZ189 during replication in human 293 cells. Mutations were induced with varying doses (4-12 microM) melphalan in the aprt gene of the hemizygous Chinese hamster ovary (CHO) cell line D422 to determine whether a similar mutation spectrum would be observed in an endogenous gene. DNA sequence alterations were determined for 39 spontaneous and 41 melphalan-induced independent mutant clones. Other than a predominance of transversions in both systems, the spectrum of melphalan-induced aprt mutations bears little resemblance to the spectrum observed in the supF gene of the shuttle plasmid pZ189. In aprt, mutations at G.C base pairs (bp) predominated (29 of 41 base substitutions). Significantly enhanced mutagenesis was observed at 5' G-G-C 3' and 5' G-G-C-C 3' sites in the aprt gene. Almost half of the melphalan-induced base substitutions occurred at 5' G-N-C 3' sequences, which are believed to be potential interstrand crosslink sites.     

Effect of cisplatin on cell death and DNA crosslinking in rat mammary. Adenocarcinoma in vitro

The pharmacodynamic effects of cisdiamminedichloroplatinum(II) (CDDP) in vitro have been reported, but the dosage and exposure time in vitro have not been based on clinical observations of the drug's actions in vivo. In this study, the authors attempted to evaluate the pharmacodynamic effects of CDDP in vitro in terms of cell survival and DNA crosslinking by simulating unbound CDDP administration at varying concentrations to a rat mammary adenocarcinoma line (known as line 66). CDDP exposure was conducted by both constant concentration procedures and a simulated in vivo procedure. Colony formation assay for the surviving fraction and alkaline elution assay for DNA crosslink measurement were performed in order to evaluate the pharmacodynamics of CDDP. Cell survival was a function of the area under the drug concentration time curve (AUC) of unbound CDDP (R2 = 0.77, P < 0.002) for all drug exposure procedures as analyzed by the analysis of covariance test. There was a strong correlation between the surviving fraction and the crosslink index of the total amount of DNA crosslinks (R2 = 0.85, P < 0.0005). Both the total amount of DNA-DNA crosslinks and the DNA-protein crosslinks, of which the latter were dominant, were affected not by the exposure procedures, but by the AUC value (P < 0.002). The thresholds of cytocidal effect were 1.59 mg.h/l for the AUC and 0.008 for the crosslink index. The pharmacodynamic effects in vitro by simulated in vivo exposure were identical to those of constant.     

Molecular nature of intrachromosomal deletions and base substitutions induced by environmental mutagens

Cellular DNA is exposed to a variety of exogenous and endogenous mutagens. A complete understanding of the importance of different types of DNA damage requires knowledge of the specific molecular alterations induced by different types of agents in specific target tissues in vivo. The gpt delta transgenic mouse model provides the opportunity to characterize tissue-specific DNA alterations because small and large deletions as well as base substitutions can be analyzed. Here, we summarize the characteristics of intrachromosomal deletions and base substitutions induced by ionizing radiation in liver and spleen, ultraviolet B (UVB) radiation in epidermis, mitomycin C (MMC) in bone marrow, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in colon, and aminophenylnorharman (APNH) in liver of gpt delta mice. Carbon-ion radiation, UVB, and MMC induced large deletions of more than 1 kb. About half of the large deletions occurred between short direct-repeat sequences and the remainder had flush ends, suggesting the involvement of nonhomologous end joining of double-stranded breaks (DSBs) in DNA. UV photoproducts and interstrand crosslinks by MMC may block DNA replication, thereby inducing DSBs. In contrast, PhIP and APNH mainly generated 1 bp deletions in runs of guanine bases. As for base substitutions, UVB and MMC induced G:C-->A:T transitions at dipyrimidine sites and tandem base substitutions at GG sites, respectively. PhIP and APNH induced G:C-->T:A transversions. Translesion DNA synthesis across the lesions, i.e., UV photoproducts, intrastrand crosslinks by MMC, and guanine adducts by the heterocyclic amines, may be involved in the induction of base substitutions. These results indicate the importance of sequence information to elucidate the mechanisms underlying deletions and base substitutions induced in vivo by environmental mutagens.     

Chromatin-associated DNA endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal DNA

The influence of nucleosome structure on the activity of 2 chromatin-associated DNA endonucleases, pIs 4.6 and 7.6, from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined on DNA containing either psoralen monoadducts or cross-links. As substrate a reconstituted nucleosomal system was utilized consisting of a plasmid DNA and either core (H2A, H2B, H3, H4), or total (core plus H1) histones from normal or XPA cells. Both non-nucleosomal and nucleosomal DNA were treated with 8-methoxypsoralen (8-MOP) plus long-wavelength ultraviolet radiation (UVA), which produces monoadducts and DNA interstrand cross-links, and angelicin plus UVA, which produces monoadducts. Both normal endonucleases were over 2-fold more active on both types of psoralen-plus-UVA-damaged core nucleosomal DNA than on damaged non-nucleosomal DNA. Addition of histone H1 to the system reduced but did not abolish this increase. By contrast, neither XPA endonuclease showed any increase on psoralen-treated nucleosomal DNA, with or without histone H1. Mixing the normal with the XPA endonucleases led to complementation of the XPA defect. These results indicate that interaction of these endonucleases with chromatin is of critical importance and that it is at this level that a defect exists in XPA endonucleases.     

Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells

Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens.     

Uranyl acetate induces hprt mutations and uranium-DNA adducts in Chinese hamster ovary EM9 cells

Questions about possible adverse health effects from exposures to uranium have arisen as a result of uranium mining, residual mine tailings and use of depleted uranium in the military. The purpose of the current study was to measure the toxicity of depleted uranium as uranyl acetate (UA) in mammalian cells. The activity of UA in the parental CHO AA8 line was compared with that in the XRCC1-deficient CHO EM9 line. Cytotoxicity was measured by clonogenic survival. A dose of 200 microM UA over 24 h produced 3.1-fold greater cell death in the CHO EM9 than the CHO AA8 line, and a dose of 300 microM was 1.7-fold more cytotoxic. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. A dose of 200 microM UA produced approximately 5-fold higher averaged induced mutant frequency in the CHO EM9 line relative to the CHO AA8 line. The generation of DNA strand breaks was measured by the alkaline comet assay at 40 min and 24 h exposures. DNA strand breaks were detected in both lines; however a dose response may have been masked by U-DNA adducts or crosslinks. Uranium-DNA adducts were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) at 24 and 48 h exposures. A maximum adduct level of 8 U atoms/10(3) DNA-P for the 300 microM dose was found in the EM9 line after 48 h. This is the first report of the formation of uranium-DNA adducts and mutations in mammalian cells after direct exposure to a depleted uranium compound. Data suggest that uranium could be chemically genotoxic and mutagenic through the formation of strand breaks and covalent U-DNA adducts. Thus the health risks for uranium exposure could go beyond those for radiation exposure.     

Functional and physical interaction between the mismatch repair and FA-BRCA pathways

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.     

Nitrogen mustard drug resistant B-cell chronic lymphocytic leukemia as an in vivo model for crosslinking agent resistance

Acquired resistance is a limiting factor in chemotherapy. We have employed nitrogen mustard resistant B-cell chronic lymphocytic leukemia (B-CLL) as a clinically relevant model to study this phenomenon. Resistance in B-CLL is associated with enhanced repair of nitrogen mustard crosslinks. In order to identify the repair pathway responsible for nitrogen mustard resistance, lymphocytes were screened for cross-resistance to a variety of DNA damaging agents. The MTT assay was used to measure the resistance of B-CLL lymphocytes to various DNA damaging agents, including nitrogen mustards, UV light, methyl methanesulfonate, and mitomycin C. We have shown that B lymphocytes from patients with nitrogen mustard resistant chronic lymphocytic leukemia reflect their clinical status. This assay allows us to classify lymphocytes as nitrogen mustard sensitive or resistant, based on in vitro observations. The resistant population was 5.6 and 4.1 fold more resistant to the nitrogen mustard analogs, chlorambucil and melphalan, respectively. Resistant lymphocytes displayed no increased resistance to either methyl methanesulfonate or UV light, indicating that neither classical base nor nucleotide excision repair is rate-limiting in resistance. Resistant lymphocytes were 6.0 and 2.2 fold more resistant to mitomycin C and cis-diamminedichloroplatinum (II), respectively, suggesting enhanced crosslink repair. Neither glutathione nor glutathione S-transferase levels correlated with resistance. The development of nitrogen mustard drug resistance in B-CLL appears to be associated with cross-resistance to other bifunctional alkylating agents which produce interstrand crosslinks. Our results indicate that resistance to nitrogen mustards in chronic lymphocytic leukemia is associated with enhanced repair of DNA crosslinks which may involve a recombination dependent system. This model should prove very useful in the elucidation of the molecular mechanisms of crosslink repair.     

Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1×10⁻² breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15–20%) after 24 h repair incubation in the tumor cell line, 5–10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1

The X-ray-sensitive mutant V79 cell line irs1 was characterized with respect to chromosomal aberrations induced by ¹³⁷Cs, mitomycin C (MMC), and decarbamoyl mitomycin C (DCMMC). To measure chromosome damage induced at different cell cycle stages, irs1 and the parental V79-4 cell lines were pulse-labeled with bromodeoxyuridine (BrdUrd) at the time of exposure and harvested at various intervals corresponding to exposure in G₁, S, and G₂ phases of the cell cycle. Metaphase spreads were stained with an anti-BrdUrd antibody, followed by a fluorescein-conjugated second antibody. With propidium iodide as a counter stain, cells were scored for aberrations. Compared to the parental V79 cells, irs1 cells had: (1) greatly increased sensitivity to all 3 agents; (2) a high frequency of chromatid exchanges after exposure in each phase of the cell cycle; and (3) more sensitivity to the agent causing crosslinks (MMC) than its monofunctional analog (DCMMC). The finding of chromatid-type damage in cells exposed to ionizing radiation during G₁ is atypical of normal cells, but is similar to observations made in several mutant rodent cell lines and in ataxia telangiectasia cells. Our results suggest that the defect in irs1 cells can manifest itself as misrepair or misreplication during all phases of the cell cycle and leads to a high incidence of chromatid exchanges and deletions.     

Xeroderma pigmentosum group a protein and chemotherapy resistance in human germ cell tumors

The exceptional sensitivity of germ cell tumors (GCTs) of adolescents and adults to chemotherapy, in particular to cisplatin, has been attributed to low levels of xeroderma pigmentosum group A protein (XPA), a crucial component of the nucleotide excision repair DNA repair pathway. In different types of solid tumors, resistance to cisplatin has been associated with enhanced expression of XPA. To assess the role of XPA levels in clinical sensitivity and resistance of GCTs to chemotherapy, immunohistochemistry was performed on tumor samples of both unselected patients before therapy and patients with fully documented clinical course before and after therapy. In the case of high XPA levels, fluorescent in situ hybridization was applied to assess the possibility of gene amplification. XPA protein levels were investigated by Western blot analysis after repeated exposure to cisplatin in different GCT-derived cell lines. Finally, XPA levels of both sensitive and cisplatin-resistant GCT cell lines were compared with cell lines derived from other neoplasms. We found that the presence of XPA protein as assessed by immunohistochemistry differs among the various histologies of GCTs. It is found more frequently and with a more homogenous staining pattern in histologic subtypes showing a more differentiated phenotype. Overall, no differences in the presence of XPA was observed between samples of tumors refractory or sensitive to chemotherapy. No XPA gene amplification was found. Interestingly, all tumors resected in relapse after chemotherapy in the refractory group stained positive for XPA. However, XPA was not induced by repeated courses of sublethal doses of cisplatin in GCT-derived cell lines in vitro, and no correlation between XPA protein levels and sensitivity to cisplatin in three GCT-derived cell lines was observed. We therefore conclude that XPA does not play a critical role in overall treatment resistance of GCTs.     

Photobiology of furocoumarins. Various types of crosslinking with DNA and their interference with the development of lambda phage

It was shown that the multiplication of phage lambda was strongly suppressed by furocoumarins after irradiation with near ultraviolet light of 365 nm wavelength. Using xanthotoxin or angelicin there was a marked inhibition of the phage DNA injection and replication but adsorption was unaffected. This inhibition was attributed to various types of DNA crosslinking produced in the phage heads. Type I crosslink corresponded to covalent binding between adjacent sites in opposite strands of the double helix. Crosslink type II (hairpin crosslink) required a highly condensed DNA and corresponded to covalent binding between adjacent sites on double-helical segments of a folded DNA molecule. The relationships of the type I crosslinks to the DNA replication and of the type II crosslinks to DNA injection are being discussed. Like type II crosslinks, the nucleic acid--protein crosslinks hinder injection.