Nucleases Specific for Ultraviolet Light-Irradiated DNA and their Possible Role in Dark Repair

185

Ultraviolet (UV)induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and UV-inactivated HVJ (Sendai virus). The present results suggest that (1) T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, (I) the enzyme was functional on human chromosomal DNA which had been damaged by UV irradiation in the viable cells, (3) all the studied groups of xeroderma pigmentosum ("variant" was not tested) were defective in the first step (incision) of excision repair. (5) suggested that XP cells fail to start the excision process because they lack the required function of an UV-specific endonuclease. Paterson et al. (6) and Buhl and Regan (7) reported that an UV-specific endonuclease from Micrococcus luteus was effective for introducing single-strand breaks in UV-damaged DNA of XP cells (both classical and deSanctis-Cacchione). On the contrary, Cleaver (8) observed that after UV-irradiation strand breaks accumulated in DNA of XP cells (deSanctis-Cacchione). All these experiments (5-8) were carried out using an alkaline-sucrose gradient centrifugation technique in vitro to estimate the degree of strand breaks of UV-irradiated DNA.We planned to identify autoradiographically what kind of cells in the five complementation groups was defective in the enzyme for strand breaks of UV-damaged DNA, by insertion of an UV-specific endonuclease into viable XP cells. HVJ is well known to have cell fusion activity (9). At an early stage of the cell fusion reaction, the structure of the Abbreviations: HVJ, hemagglutirating virus of Japan, synonym: Sendai virus; XP, xeroderma pigmentosum; UV, ultraviolet; PEME, phosphate-ethylene glycol-mercaptoethanol-EDTA buffer; ENase, endonuclease V. * To whom all correspondence should be addressed. 4071cell membrane is partially damaged by the action of the virus and then the structure is restored again. Thus, it seemed possible that macromolecules could be inserted into viable cells from the outside during the interaction between the cell membrane and HVJ. As an enzyme, T4 endonuclease V was used, which was isolated from Escherichia coli infected with bacteriophage T4 (10,11). This enzyme catalyzes the first step of excision repair in T4-infected cells (12). We tried to insert this enzyme into XP cells with help of HVJ, and this trial was successful. MATERIALS AND METHODSCells. Human fibroblasts derived from biopsy specimens of skin of normal subjects and XP patients were grown as monolayers in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (Flow Lab.). Strain HNSF3 was derived from normal skin. Strains XP80S, XP1OOS, and XP6TOO were from Japanese XP patients and have been classified in complementation group A §. Strain CRL 1228, 1199, 1170, 1160, and 1259, belonging to the complementation groups A, B, C, D, and E, respectively, were obtain...

Section: Discussionmentioning

confidence: 86%

Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus).

Tanaka¹,

Sekiguchi²,

Okada³

1975

185

“…These activities are absent from some UV-sensitive mutants (8,10). Takagi et al, have described an activity in extracts of E. coli that specifically incises UV-irradiated DNA (11). However, since this activity was reported to be present in extracts of all studied U1-sensitive mutants, the role of this enzymatic activity in excision repair was not clear.…”

mentioning

confidence: 99%

An Endonuclease from Escherichia coli That Acts Preferentially on UV-Irradiated DNA and Is Absent from the uvrA and uvrB Mutants

Braun

Grossman

1974

125

At least two endonucleolytic activities that preferentially incise ultraviolet (UV)-irradiated DNA exist in extracts of E. coli. These two activities can be separated by phosphocellulose chromatographic fractionation. The subject of this paper is one of these activities, which elutes from phosphocellulose with 0.25 M potassium phosphate, pH 7.5. This endonucleolytic activity specific for UV-irradiated DNA is absent from partially purified extracts of uvrA and uvrB mutants, which are defective in excision of pyrimidine dimers, but is present in normal amounts in the uvrC excision-defective mutant. The enzyme binds very tightly and specifically to UV-irradiated DNA. Binding can be prevented by prior treatment of the irradiated DNA with photoreactivating enzyme. This binding activity is absent in uvrA and uvrB mutants, but present in uvrC and uvrD mutants.Excision repair of UV-induced cyclobutane-type pyrimidine dimers in Escherichia coli is currently thought to be a fourstage process (1). Initially, an endonucleolytic incision is made in the vicinity of the dimer. This is followed by exonucleolytic removal of the dimer and several adjacent nucleotides. Nucleotides are reinserted into the resultant cavity by a polymerase, using the opposite, intact strand as a template, with the continuity of the repaired region restored by polynucleotide ligase.Mutants of E. coli with markedly reduced levels of polymerase I (2) and polynucleotide ligase (3, 4) are substantially more UV-sensitive than the wild type. UvrA, uvrB, and uvrC mutants do not excise dimers (5) and presumably are defective in either the endonucleolytic or exonucleolytic steps of excision repair.Endonucleolytic activities specific for UV-irradiated DNA have been isolated and purified from Micrococcus luteus (6, 7) and E. coli infected with phage T4 (8, 9). These activities are absent from some UV-sensitive mutants (8, 10). Takagi et al., have described an activity in extracts of E. coli that specifically incises UV-irradiated DNA (11). However, since this activity was reported to be present in extracts of all studied U1-sensitive mutants, the role of this enzymatic activity in excision repair was not clear.In this paper, we describe the partial purification and some of the properties of an endonucleolytic activity, specific was prepared by methods described by Schekman et al. (12). This method involves ethidium bromide-CsCl density gradient centrifugation followed by a neutral sucrose gradient. A substantial amount of material absorbing 260-nm light, presumably unlabeled RNA, is removed in the sucrose gradient. In many of the preparations used in these experiments, the final gradient was omitted. This omission had no detectable effect on the assays described below.UV-Irradiation. The OX RFI ['H]DNA was irradiated in a stoppered quartz cuvette at 280 nm with a monochrometer described previously (6). In general, irradiation was to an average dose of 9000 ergs/mm2. The incident dose was adjusted to compensate for self-absorption.Enzymes. M. luteus UV-en...

“…A number of UV-sensitive strains from E. coli and from M. luteus are unable to excise dimers after UV-irradiation and show severely restricted reactivation of UV-damaged bacteriophage. When assayed in vitro for the presence of UV-specific endonuclease, these strains have normal levels of enzyme activity (35,36), while a mutant from Ml. luteus lacking this UV-specific endonuclease is only slightly UV-sensitive (36,37).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Excision-Repair of Ultraviolet-Irradiated Transforming DNA from Bacillus subtilis

Heijneker

Pannekoek

Oosterbaan

et al. 1971

The biological activity of UV-inactivated Bacillus subtilis DNA is partly restored after incubation with a UV-specific endonuclease from Micrococcus luteus in conjunction with DNA polymerase and DNA ligase, both isolated from Escherichia coli. The restored activity is not further increased by photoreactivation. Pyrimidine dimers are specifically liberated when irradiated DNA is exposed to the three enzymes. None of these effects is observed when pancreatic DNase is used instead of UV-specific endonuclease.Since the demonstration by RMrsch et al. (1) and by Elder and Beers (2) that dark-repair of UV-irradiated DNA is enzymatic in nature, much effort has been given to elucidate its mechanism. Using UV-irradiated OX 174 replicative form type I (RFI) DNA and extracts from Micrococcus luteus, Rorsch et al. (3) showed that simultaneously with an increase in biological activity (assayed as the ability to form virus particles in spheroplasts of Escherichia coli), single-strand breaks are introduced in RFI-DNA, suggesting that an endonuclease is involved in the reactivation process.Setlow and Carrier (4) -and Boyce and Howard-Flanders (5) observed that thymine dimers are excised from UV-irradiated DNA in the in vivo dark-repair process. They proposed a repair mechanism in which dimers are removed by the combined action of an endonuclease, which recognizes dimers, and an exonuclease, which excises the UV-damaged region. After repair-replication (6), the physical continuity of the DNA strand is restored by DNA ligase (7).Results of in vitro experiments support this model: Carrier and Setlow (8) were able to demonstrate excision of pyrimidine dimers from UV-irradiated DNA in extracts from M. luteus. Both a UV-specific endonuclease and an exonuclease have been purified from this organism (9, 10). The endonuclease specifically recognizes dimers (11,12). Recently, Kelly et al. (13) demonstrated that DNA polymerase from E. coli is able to excise pyrimidine dimers and to insert new nucleotides into the DNA chain in the 5' to 3' direction, thus 'translating' single-strand nicks.These results prompted us to investigate whether UV-irradiated DNA can be repaired in vitro with UV-specific endonuclease, DNA polymerase, and DNA ligase. To detect repair, we measured the recovery of biological activity of UVinactivated Bacillus subtilis transforming DNA.The B. subtilis transformation system is particularly well suited for this purpose. First, mutants of B. subtilis are known in which in vivo dark-repair of UV-inactivated transforming DNA is blocked (14) §, so that in vitro repair of UV-damage is not obscured by repair occurring in vivo. Second, in UVsensitive mutants of B. subtilis, biological activity of UVinactivated transforming DNA is further decreased after it is nicked with UV-specific endonuclease (14), implying that complete dark-repair would have to occur in vitro before an increase in biological activity could be observed. This is in contrast with other biological test systems, such as Haemophilus influenzae transforma...