Crude Extracts Of HeLa Cells Research Articles

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

High‐performance affinity chromatography method for identification of L‐arginine interacting factors using magnetic nanobeads

L-Arginine exhibits a wide range of biological activities through a complex and highly regulated set of pathways that remain incompletely understood at both the whole-body and the cellular levels. The aim of this study is to develop and validate effective purification system for L-arginine interacting factors (AIFs). We have recently developed novel magnetic nanobeads (FG beads) composed of magnetite particles/glycidyl methacrylate (GMA)-styrene copolymer/covered GMA. These nanobeads have shown higher performance compared with commercially available magnetic beads in terms of purification efficiency. In this study, we have newly developed L-arginine methyl ester (L-AME)-immobilized beads by conjugating L-AME to the surface of these nanobeads. Firstly, we showed that inducible nitric oxide synthase, which binds and uses L-arginine as a substrate, specifically bound to L-AME-immobilized beads. Secondly, we newly identified phosphofructokinase, RuvB-like 1 and RuvB-like 2 as AIFs from crude extracts of HeLa cells using this affinity chromatographic system. The data presented here demonstrate that L-AME-immobilized beads are effective tool for purification of AIFs directly from crude cell extracts. We expect that the present method can be used to purify AIFs from various types of cells.

A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing

Dynamic modification of proteins with the small ubiquitin-like modifier (SUMO) affects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro fluorescence-resonance-energy-transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that differential maturation rates of SUMO paralogs in vivo may be functionally significant. The high degree of structural conservation across the ubiquitin-like protein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modification systems.

NAP-2: histone chaperone function and phosphorylation state through the cell cycle

We have recently cloned the human nucleosome assembly protein 2 (NAP-2). Here, we demonstrate that casein kinase 2 (CKII) from HeLa cell nuclear extracts interacts with immobilized NAP-II, and phosphorylates both NAP-2 and nucleosome assembly protein 1 (NAP-1) in vitro. Furthermore, NAP-1 and NAP-2 phosphorylation in crude HeLa cell extracts is abolished by heparin, a specific inhibitor of CKII. Addition of core histones can stimulate phosphorylation of NAP-1 and NAP-2 by CKII. NAP-2 is also a phosphoprotein in vivo. The protein is phosphorylated at the G0/G1 boundary but it is not phosphorylated in S-phase. Here, we show that NAP-2 is a histone chaperone throughout the cell cycle and that its cell-cycle distribution might be governed by its phosphorylation status. Phosphorylated NAP-2 remains in the cytoplasm in a complex with histones during the G0/G1 transition, whereas its dephosphorylation triggers its transport into the nucleus, at the G1/S-boundary, with the histone cargo, suggesting that binding to histones does not depend on phosphorylation status. Finally, indirect immunofluorescence shows that NAP-2 is present during metaphase of HeLa and COS cells, and its localization is distinct from metaphase chromosomes.

The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli.

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.

Further purification and characterization of a multienzyme complex for DNA synthesis in human cells

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems.

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.

DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5′ → 3′ exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.

The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats

I have identified an activity in crude HeLa cell extracts that satisfies the requirements for a human telomere terminal transferase or telomerase. It catalyzes the addition of a 6 nucleotide repeating pattern to oligonucleotide primers containing human or nonhuman telomeric repeat sequences. Direct sequence analyses of reaction products reveal the added sequence to be TTAGGG in all cases. Under optimal conditions 65–70 repeats can be synthesized. The enzyme has the properties of a ribonucleoprotein. Telomerase has previously been observed only in ciliated protozoans, which possess 104–107 macronuclear telomeres. The identification of telomerase in HeLa cells with only ∼100 telomeres indicates that telomerase-mediated telomere maintenance is conserved throughout eukaryotes.

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots.

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.

Lack of excision of 4HAQO adducts from DNA by cell extracts that excise pyrimidine dimers

A substrate of DNA containing 4HAQO adducts, suitable for studies of excision repair, was prepared by reacting calf thymus DNA with [ 3H]-monoacetyl-4HAQO. A crude HeLa cell extract was prepared by the method of Mortelmans et al (Proc. Natl. Acad. Sci. U.S.A. 73 , 2757, 1976). The cell extract would specifically excise pyrimidine dimers from UV-irradiated DNA but would not release 4HAQO adducts in an acid soluble form. This result points to different initial steps in the excision repair process for these two forms of damage even though much of the repair mechanism is common to both.

Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.

Initiation and regulation of simian virus 40 early transcription in vitro.

We have studied initiation and regulation of early transcription of simian virus (SV40) DNA in vitro by eucaryotic RNA polymerase II, using both a crude HeLa cell extract and a partially purified calf thymus polymerase supplemented with a HeLa cell S100 fraction. Analysis of initiation sites by primer-directed cDNA synthesis and sequencing of cDNA's has revealed that early transcription is initiated at a multiplicity of sites corresponding to the 5' termini of early viral mRNA's. The pattern of in vitro initiation closely resembles the pattern of 5' termini of early mRNA's late in the lytic cycle, with principal initiations between residues 5184 to 5194, upstream from the early Hogness-Goldberg (TATA) sequence, and at residue 5123, well downstream from this sequence. In vitro transcription is initiated to a lesser extent at sites between residues 5150 and 5155, the principal 5' termini of early mRNA's in transformed cells and early in lytic infection, located 21 to 26 nucleotides downstream from the TATA sequence. Initiation occurs at identical sites and with similar efficiencies on form I and linearized DNA templates. There are minor differences in the efficiency of initiation at specific sites by the two transcriptional systems. Studies using a DNA template cleaved just downstream from the TATA sequence and a second template cleaved through a pair of 72-base-pair tandem repeats starting 87 nucleotides upstream from the TATA sequence have revealed that neither the TATA sequence nor the repeats are essential for early transcription in vitro. However, removal of the TATA and upstream sequences shifts initiation of transcription principally to the residue 5123 site. Comparison of the relative efficiencies of transcription on intact wild-type DNA, the two cleaved DNAs, and DNA from a deletion mutant suggests that all or most of the sequences constituting an early promoter lie within the genomic region 60-70 to 140 nucleotides upstream from the principal 5' termini of the early mRNA's.

Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules.

One of the major groups of microtubule-associated proteins (MAPs) found associated with the microtubules isolated from HeLa cells has a molecular weight of just over 200,000. Previous work has demonstrated that these heLa MAPs are similar in several properties to MAP-2, one of the major MAPs of mammalian neural microtubules, although the two types of proteins are immunologically distinct. The 200,000 mol wt HeLa MAPs have now been found to remain soluble after incubation in a boiling water bath and to retain the ability to promote tubulin polymerization after this treatment, two unusual properties also shown by neural MAP-2. This property of heat stability has allowed the development of a simplified procedure for purification of the 200,000 HeLa MAPs and has provided a means for detection of these proteins, even in crude cell extracts. These studies have also led to the detection of a protein in crude extracts of HeLa cells and in cycled HeLa microtubules which has been identified as MAP-2 on the basis of (a) comigration with calf brain MAP-2 on SDS PAGE, (b) presence in purified microtubules, (c) heat stability, and (d) reaction with two types of antibodies prepared against neural high molecular weight-MAPs, one of these a monoclonal antibody against hog brain MAP-2, although present in HeLa cells, is at all stages of microtubule purification a relatively minor component in comparison to the 200,000 HeLa MAP's.

Human endonuclease activity for DNA apurinic sites.

APURINIC sites in cellular DNA may occur secondary to the slow hydrolysis of purines in physiological conditions1 or the rapid hydrolysis of alkylated purines2. Endonucleases which incise double-stranded DNA at apurinic sites have been purified from E. coli3, calf thymus4 and rat liver5. It has been proposed that such endonucleases hydrolyse the apurinic site as the first step of a repair process similar to excision-repair of ultraviolet photoproducts6. Lindahl and Nyberg calculated that spontaneous depurination, if unrepaired in postmitotic cells such as human neurones, would result in the loss of 3% of the total purines during a lifetime1. Such a loss could be sufficient to produce abnormal proteins which appear in ageing cells7. The more rapid depurination of alkylated bases could be related to the mutagenic and carcinogenic properties of alkylating agents8. Therefore, to relate the repair of apurinic sites to the pathogenesis of human disease, we have characterised the properties of apurinic site endonuclease activity in crude extracts of HeLa cells and then measured such activity in WI-38 cells and human skin fibroblasts. We then compared enzyme activity in skin fibroblasts from individuals with two diseases in which repair of apurinic sites might be defective—progeria, characterised by premature ageing9 and associated with abnormal thermolability of cellular enzymes10, and Fanconi's anaemia, associated with an increased incidence of malignancy and in vitro susceptibility to chromosome breakage by alkylating agents11.

DNA-degradation and excision repair in gamma-irradiated Chinese hamster ovary cells.

THE removal from the DNA of cyclobutane-type photo-dimers following exposure to ultraviolet irradiation has been demonstrated in human cells1,2. No excision of photo-dimers has, however, been observed in rodent cells3–6 or in cells from patients with the hereditary disease xeroderma pigmentosum2,7. Recently, it has been recognised that damage to the nucleic acid bases may also constitute a major portion of the damage introduced into DNA by ionising radiation (refs 8, 9 and J. L. Swinehart, W.-S. Lin, J. Roti Roti, R. B. Painter and P. A. C., unpublished). The following evidence suggests that excision repair processes may occur following exposure to ionising radiation. The insertion of short patches of nucleotides into the DNA in γ-irradiated mammalian cells has been observed in the form of repair replication and unscheduled DNA synthesis10. These processes are best understood in molecular terms if it is assumed that damaged and undamaged residues are first removed from the DNA in early steps of postirradiation repair. Endonuclease activities which appear to recognise γ-ray-induced nucleotide damage in in vitro systems have recently been identified. A partially purified preparation from Micrococcus luteus has been studied by Carrier and Setlow11 and an endonuclease activity in crude HeLa cell extracts by Brent12. Such enzyme activities could be involved in the excision repair of γ-ray damaged residues. We report here that Chinese hamster ovary cells (CHO) undergo limited DNA degradation following exposure to γ rays and we demonstrate by direct chemical analysis the excision of damaged thymine residues from the DNA during post-radiation incubation.

Ultraviolet-induced changes in DNA: Possible confusion of repair and degradative enzymes

It has recently been reported (Burt, D. H. and Brent, T. P. (1971) Biochem. Biophys. Res. Commun. 43, 1382–1387) that a deoxyribonuclease specific for ultraviolet-irradiated DNA is found in crude extracts of HeLa cells. We find a similar deoxyribonuclease activity in both normal and xeroderma pigmentosum cells, which indicates that the activity is not correlated with the defective excision repair in xeroderma pigmentosum cells. This raises the possibility that the deoxyribonuclease activity is not really specific for ultraviolet-irradiated DNA. Our results show that irradiation of DNA with extremely high doses produces extensive breaks and partial denaturation of the DNA. Use of DNA irradiated with high ultraviolet doses as a substrate for deoxyribonucleases detected an enzyme which degraded the single-stranded regions of DNA rather than attacking the ultraviolet photoproducts. The enzyme was partially purified by electrophoresis and was in fact found to be an exonuclease specific for single-stranded DNA.