Two DNA endonuclease activities from normal human and xeroderma pigmentosum chromatin active on psoralen plus ultraviolet light treated DNA

DNA endonuclease activities from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were examined on DNA treated with 8-methoxypsoralen (8-MOP) or 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light, which produce monoadducts and DNA interstrand cross-links, and angelicin plus UVA light, which produces mainly monoadducts. 9 chromatin-associated DNA endonuclease activities were isolated from normal and XPA cells and assayed for activity on PM2 bacteriophage DNA that had been treated with 8-MOP or TMP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given. Cross-linking of DNA molecules was confirmed by alkaline gel electrophoresis. In both normal and XPA cells, two DNA endonuclease activities were found which were active on 8-MOP and TMP plus UVA light treated DNA. One of these endonuclease activities, pI 4.6, is also active on intercalated DNA and a second one, pI 7.6, is also active on UVC (254 nm) light irradiated DNA. The major activity against angelicin plus UVA light treated DNA in both normal and XPA cells was found in the fraction, pI 7.6. The levels of activity of both of these fractions on all 3 psoralen-damaged DNAs were similar between normal and XPA cells. These results indicate that in both normal and XPA cells there are at least two different DNA endonucleases which act on both 8-MOP and TMP plus UVA light treated DNA.     

A damage-recognition protein which binds to DNA containing interstrand cross-links is absent or defective in Fanconi anemia, complementation group A, cells.

A DNA binding protein with specificity for DNA containing interstrand cross-links induced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light has been identified in normal human chromatin. Protein binding to DNA was determined using a gel mobility shift assay and an oligonucleotide containing a hot spot for formation of psoralen interstrand cross-links. Specificity of the damage-recognition protein for cross-links was demonstrated both by a positive correlation between level of cross-link formation in DNA and extent of protein binding and by effective competition by treated but not undamaged DNA for the binding protein. Chromatin protein extracts from cells from individuals with the genetic disorder, Fanconi anemia, complementation group A (FA-A), which have decreased ability to repair damage produced by TMP plus UVA light, failed to show any protein binding to TMP plus UVA treated DNA. We have previously shown that these chromatin protein extracts contain a DNA endonuclease complex, pI 4.6, which specifically recognizes and incises DNA containing interstrand cross-links and which in FA-A cells is defective in its ability to incise this damaged DNA (Lambert et al. (1992) Mutation Res., 273, 57-71). Together, these findings suggest that the DNA binding protein identified is involved in recognition and repair of DNA interstrand cross-links.     

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Can a mixed damage interfere with DNA‐prottein cross‐links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo , by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.     

Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.     

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells

Summary Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5,8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7–2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost neglible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan     

Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: The RAD3 system and the RAD51 system

Summary We have studied the role of the excision-repair system and the recombination-repair system in the removal of cross-links and monoadducts caused by furocoumarins plus 360 nm radiation in yeast DNA by neutral and alkaline sucrose gradients and by a fluorometric procedure which detects cross-linked DNA molecules. We found that the excision-repair system, represented by the rad3 mutations, is required both for the removal of monoadducts, causing single-strand break formation, and for the removal of cross-links, causing double-strand break formation. The recombination-repair system, represented by the rad51 mutation, is necessary for double-strand break repair following cross-link removal, but it has no role in the repair of monoadducts.It can be concluded that at least some of the same enzymes are used in yeast for both the excision of pyrimidine dimers and the excision of cross-links or monoadducts caused by furocoumarins plus light. The RAD3 and RAD51 repair systems, which act independently in the repair of UV-induced lesions, are part of a single system for the repair of cross-links.     

Inactivation of Escherichia coli, F′ Episomes at Transfer, and Bacteriophage Lambda by Psoralen Plus 360-nm Light: Significance of Deoxyribonucleic Acid Cross-Links

We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac(+) episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr(-) strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.     

Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.     

Genetic control of the bypass of mono-adducts and of the repair of cross-links photoinduced by 8-methoxypsoralen in yeast

A large UVA dose by itself induces lethal damage revealed in some repair-deficient strains of Saccharomyces cerevisiae. Following photoaddition of a monofunctional psoralen derivative, 3-carbethoxypsoralen, an extra killing effect is observed by applying a second high UVA dose, in conditions where a fraction of 8-methoxypsoralen (8-MOP) plus UVA-induced monoadducts are transformed into DNA cross-links. In an excision-repair-deficient context, the bypass of 8-MOP plus UVA-induced monoadducts is under the control of the RAD6+ gene product. However, when other steps of the mutagenic pathway are blocked by the rad18-2 or the pso1-1 mutations, bypass occurs. This is also true when in excision-deficient strains the recombinogenic pathway is blocked by the rad52-1 mutation. The recombinogenic pathway may be an alternative to the mutagenic pathway for bypass of monoadducts. The repair of the lesions induced by a second UVA dose applied after a first treatment by 8-MOP plus UVA [i.e. cross-links and other putative lesion(s)] is controlled by at least the RAD2+, RAD6+, RAD52+, PSO2+ and PSO1+ gene products. The role of the pathways involved is discussed according to the nature of the secondarily induced lesions.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

DNA semi-conservative synthesis in normal and Fanconi anemia fibroblasts following treatment with 8-methoxypsoralen and near ultraviolet light or with X-rays

Summary The effect of treatment with 8-methoxypsoralen (8-MOP) plus near-UV radiation (UVA) or with X-rays on the rate of DNA semi-conservative synthesis of fibroblasts from 10 Fanconi anemia (FA), two heterozygous, and three normal cell lines was studied. Following treatments with either X-rays or low doses of 8-MOP plus UVA leading to a majority of monoadducts over cross-links per genome, the FA and hetcrozygous cell lines were indistinguishable from normals: the transient inhibition of semi-conservative DNA synthesis was followed by the recovery of a nomral rate of synthesis. In contrast treatment with higher (but not saturating) doses of 8-MOP plus UVA allowed us to distinguish two classes among the FA cell lines. One class demonstrated a pattern of recovery similar to that of heterozygous and normal cell lines. This indicates that in such cell lines, the predominant lesion in this condition, the cross-links, do not arrest DNA synthesis and are likely to be normally repaired. Another class of FA cell lines did not show a recovery of a normal rate of DNA synthesis even after prolonged post-treatment incubation and although the proportion of cells in S phase was similar to that of the strains of the first category. This indicates that in such cell lines the repair of cross-links is inhibited at some step which is not necessarily the incision one.     

Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.     

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.     

Defective DNA endonuclease activities in Fanconi's anemia cells, complementation groups A and B.

Cells from patients with the inherited disorder, Fanconi's anemia (FA), were analyzed for endonucleases which recognize DNA interstrand cross-links and monoadducts produced by psoralen plus UVA irradiation. Two chromatin-associated DNA endonuclease activities, defective in their ability to incise DNA-containing adducts produced by psoralen plus UVA light, have been identified and isolated in nuclei of FA cells. In FA complementation group A (FA-A) cells, one endonuclease activity, pI 4.6, which recognizes psoralen intercalation and interstrand cross-links, has 25% of the activity of the normal human endonuclease, pI 4.6, on 8-methoxypsoralen (8-MOP) plus UVA-damaged DNA. In FA complementation group B (FA-B) cells, a second endonuclease activity, pI 7.6, which recognizes psoralen monoadducts, has 50% and 55% of the activity, respectively, of the corresponding normal endonuclease on 8-MOP or angelicin plus UVA-damaged DNA. Kinetic analysis reveals that both the FA-A endonuclease activity, pI 4.6, and the FA-B endonuclease activity, pI 7.6, have decreased affinity for psoralen plus UVA-damaged DNA. Both the normal and FA endonucleases showed approximately a 2.5-fold increase in activity on psoralen plus UVA-damaged reconstituted nucleosomal DNA compared to damaged non-nucleosomal DNA, indicating that interaction of these FA endonucleases with nucleosomal DNA is not impaired. These deficiencies in two nuclear DNA endonuclease activities from FA-A and FA-B cells correlate with decreased levels of unscheduled DNA synthesis (UDS), in response to 8-MOP or angelicin plus UVA irradiation, in these cells in culture.