Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase

Previously it had been shown that poly(ADP-ribose) polymerase requires DNA for its activity and that this enzyme is auto-poly(ADP-ribosyl)ated. The studies reported here indicate that this self-modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP-ribose) polymerase with poly(ADP-ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP-ribose) and restoring the polymerase-DNA complex. The assay of polymerase in the presence of glyco-hydrolase was made possible by use of a double-label assay involving release of 14C-labelled nicotinamide and the incorporation of 3H-labelled ADP-ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone H1 appear to activate the polymerase by increasing the affinity of the polymerase for DNA.     

Measurement of poly(ADP-ribose) glycohydrolase activity by high resolution polyacrylamide gel electrophoresis: Specific inhibition by histones and nuclear matrix proteins

We have developed a novel enzyme assay that allows the simultaneous determination of noncovalent interactions of poly(ADP-ribose) with nuclear proteins as well as poly(ADP-ribose) glycohydrolase (PARG) activity by high resolution polyacrylamide gel electrophoresis. ADP-ribose chains between 2 and 70 residues in size were enzymatically synthesized with pure poly(ADP-ribose) polymerase (PARP) and were purified by affinity chromatography on a boronate resin following alkaline release from protein. This preparation of polymers of ADP-ribose was used as the enzyme substrate for purified PARG. We also obtained the nuclear matrix fraction from rat liver nuclei and measured the enzyme activity of purified PARG in the presence or absence of either histone proteins or nuclear matrix proteins. Both resulted in a marked inhibition of PARG activity as determined by the decrease in the formation of monomeric ADP-ribose. The inhibition of PARG was presumably due to the non-covalent interactions of these proteins with free ADP-ribose polymers. Thus, the presence of histone and nuclear matrix proteins should be taken into consideration when measuring PARG activity.     

Effect of DNA on poly (ADP-ribose) glycohydrolase and the degradation of histone H1-poly (ADP-ribose) complex from HeLa cell nuclei.

A poly(ADP-ribose)-H1 histone complex has been isolated from HeLa cell nuclei incubated with NAD. The rate of poly(ADP-ribose) glycohydrolase catalyzed hydrolysis of the polymer in the complex is only 1/9 that of free poly(ADP-ribose), indicating that the polymer is in a protected environment within the complex. Comparison of the rate of hydrolysis of free poly(ADP-ribose) in the presence or absence of H1 to that in the complex synthesized de novo indicates a specific mode of packaging of the complex. This is further indicated by the fact that alkaline dissociation of the complex followed by neutralization markedly exposes the associated poly(ADP-ribose) to the glycohydrolase. The complex also partially unfolds when it binds to DNA as evidenced by a 2-fold increase in the rate of glycolytic cleavage of poly(ADP-ribose). This effect of DNA is not due to a stimulation of the glycohydrolase per se since hydrolysis of free polymer by the enzyme is strongly inhibited by DNA, especially single-stranded DNA. Inhibition of glycohydrolase by DNA results from the binding of the enzyme to DNA and conditions which decrease this binding (increased ionic strength or addition of histone H1 which competes for DNA binding) relieve the DNA inhibition.     

Purification and properties of an (ADP-ribose)n glycohydrolase from guinea pig liver nuclei

An (ADP-ribose)n glycohydrolase has been purified more than 3,000-fold from guinea pig liver nuclei with an 18% yield. The glycohydrolase activity present in the nuclei was solubilized only by sonication at high ionic strength and purified by sequential chromatographic steps on phosphocellulose, DEAE-cellulose, Blue Sepharose, and single-stranded DNA cellulose. The purified protein exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 75,500. On Sephadex G-100 gel filtration, single coincident peaks of (ADP-ribose)n glycohydrolase activity and protein with a molecular weight value of 72,000 were observed. The Km value for (ADP-ribose)n and the maximal velocity of the highly purified glycohydrolase were 2.3 microM and 36 mumol of ADP-ribose released from (ADP-ribose)n . min-1 . mg protein-1, respectively. Hydrolysis of (ADP-ribose)n by the enzyme was exoglycosidic in nature. The optimum pH for the enzyme activity was apparent at 6.8-7.0. Sulfhydryl compounds and monovalent cations were required for the maximal activity. The enzyme was sensitive to Ca2+ but not to Mg2+. The enzyme activity was inhibited by ADP-ribose, cyclic AMP (adenosine 3':5'-monophosphate) and diadenosine 5',5'-p1,p4-tetraphosphate. Denatured DNA and histones were inhibitory, but native DNA and its histone complex were not inhibitory. Our data indicate that the glycohydrolase is present only as a minor protein in nuclei, being present in perhaps about 50,000 molecules/nucleus.     

Tannins elevate the level of poly(ADP-ribose) in HeLa cell extracts

Phenolic phytochemicals such as tannins, which are natural constituents of green tea, red wine, and other plant products, are considered to have cancer-preventive properties. An important endogenous mediator of tumorigenesis is the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 synthesizes polymers of ADP-ribose (PAR), which, in turn, are degraded by the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG). In the present study, we investigated the effects of tannins on the level of PAR in HeLa nuclear extracts. The addition of tannins to nuclear extracts led to a 40-fold elevation of PAR-levels. The observed increased PAR-levels resulted from inhibition of the catalytic activity of PARG. Additionally, the human PARG cDNA was cloned and the recombinant enzyme was overexpressed and isolated. Recombinant PARG was immobilized using an affinity column composed of tannins covalently linked to Sepharose beads. Finally, an interaction between immobilized PARG and endogenous PARP-1 from HeLa cell extracts is demonstrated.     

Rapid assay of poly(ADP-ribose) glycohydrolase

We have developed a rapid, highly reproducible assay to determine poly(ADP-ribose) glycohydrolase activity which measures directly the appearance of the reaction product. We also analysed the majority of different techniques which are used to determine poly(ADP-ribose) glycohydrolase activity and found that the apparent activity can vary extensively depending on the method used. Thin-layer chromatography using PEI-F-cellulose was the only method which evaluated directly the specific release of ADP-ribose; by comparison with this method, the other procedures gave an over- or under-estimation of 2- to 10-fold of the enzymatic activity. A rapid method of affinity chromatography has also been developed to synthesize and purify in high yield poly(ADP-ribose) (35% conversion of 1 mM NAD to poly(ADP-ribose)).     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Change in activity of nuclear poly(ADP-ribose) glycohydrolase during the HeLa S3 cell cycle

The change in activity of nuclear poly(ADP-ribose) glycohydrolase during the cell cycle of HeLa S3 cells was investigated. The poly(ADP-ribose) glycohydrolase activity was solubilized from HeLa S3 cell nuclei and chromosomes only by sonication at high ionic strength. The enzyme hydrolyzed poly(ADP-ribose) exoglycosidically, producing ADP-ribose. After release from mitosis, the activity of the solubilized nuclear poly(ADP-ribose) glycohydrolase per nucleus or per unit protein, assayed with [3H]poly(ADP-ribose) (average chain length, n = 15) as substrate, was lowest in the early G1 phase and highest in the late G1 phase. The specific activity in the late G1 phase was about two times that in the early G1 phase. The high activity remained constant during the S-G2-M phase. A similar change during the cell cycle was observed after release from hydroxyurea block. These results suggest that the activity of poly(ADP-ribose) glycohydrolase doubled during the G1 phase of the cell cycle of HeLa S3 cells.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Poly(ADP-ribose) degradation by glycohydrolase starts with an endonucleolytic incision

We have recently shown that poly(ADP-ribose) polymerase forms poly(ADP-ribose) by adding ADP-ribose residues to the polymerase-proximal end of an enzyme-bound nascent chain. In this light we have reexamined the mode of hydrolysis of enzyme-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. When the substrate has been labeled by a pulse-chase protocol, soluble glycohydrolase releases a significant amount of labeled oligomer which can only come from the enzyme-distal (2') end of the polymer. This constitutes additional evidence for the proximal growth of chains. Oligomer is infrequently released from the proximal (1") end of enzyme-bound chains. Rather, the bulk of the poly(ADP-ribose) is digested directly to ADP-ribose monomers. We conclude that poly(ADP-ribose) glycohydrolase starts digestion with an endonucleolytic incision and then removes ADP-ribose residues processively in the 2'----1" direction. Therefore, in contrast to earlier models of polymer growth and hydrolysis, a single poly(ADP-ribose) chain may be extended at one end and simultaneously degraded at the other end. The balance between synthesis and degradation may control the quantity and distribution of polymer around the DNA break which occasions its synthesis.     

Poly(ADP-ribose) glycohydrolase is present in metaphase chromosomes of HeLa S3 cells

Poly(ADP-ribose) glycohydrolase was found in metaphase chromosomes of HeLa S3 cells. Adenosine diphosphate ribose and 3′, 5′-cyclic AMP inhibited the glycohydrolase activity, whereas ADP, ATP, NAD and 3′,5′-cyclic GMP did not. The hydrolytic product of poly(ADP-ribose) bound to metaphase chromosomes with this enzyme was identified as adenosine diphosphate ribose.     

Purification of acetyllactosamine-specific tomato lectin by erythroglycan-sepharose affinity chromatography

Tomato lectin is specific for oligomers of poly-N-acetyllactosamine containing 3 repeating Gal(beta 1-4)GlcNAc (beta 1-3)-disaccharides. As such it is highly useful for purifying oligosaccharides or glycopeptides with poly-N-acetyllactosamine character. We have found the lectin very useful as an affinity reagent for isolating glycoproteins or glycoprotein domains having poly-N-acetyllactosamine glycosylation. Conventional preparation of tomato lectin by ovomucoid-Sepharose affinity chromatography was found to be unsatisfactory due to instability of column and bleeding of ovomucoid into eluents requiring the necessity for additional purification steps following affinity chromatography. We prepared a column of human erythrocyte band 3 carbohydrate glycopeptide (erythroglycan) attached to Sepharose as an affinity matrix. The purification of tomato lectin to homogeneity in one step on this column matrix is described in this report.     

Evidence for an inhibitory effect exerted by yeast NMN adenylyltransferase on poly(ADP-ribose) polymerase activity

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.     

Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study.

A rapid method of purifying snake venom phosphodiesterase has been developed using Blue Sepharose or blue dextran/Sepharose as an affinity adsorbent. A sixty-fold purification of the enzyme from commercial preparations is achieved in a single step with a yield of 60%. The purified enzyme preparation is essentially free from phosphatase activities and exhibits a major protein band on SDS-polyacrylamide gel electrophoresis. Chain length analysis of poly(ADP-ribose) exemplifies the usefulness of this technique.     

Characterization of two forms of poly(ADP-ribose) glycohydrolase in guinea pig liver

A poly(ADP-ribose) glycohydrolase from guinea pig liver cytoplasm has been purified approximately 45,000-fold to apparent homogeneity. The cytoplasmic poly(ADP-ribose) glycohydrolase designated form II differed in several respects from the nuclear poly(ADP-ribose) glycohydrolase I (Mr = 75,500) previously purified from the same tissue (Tanuma et al., 1986a). The purified glycohydrolase II consists of a single polypeptide with Mr of 59,500 estimated by a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 57,000 was determined by gel permeation. Peptide analysis of partial proteolytic degradation of glycohydrolases II and I with Staphylococcus aureus V8 protease revealed that the two enzymes were structurally different. Amino acid analysis showed that glycohydrolase II had a relatively low proportion of basic amino acid residues as compared with glycohydrolase I. Glycohydrolase II and I were acidic proteins with isoelectric points of 6.2 and 6.6, respectively. The optimum pH for glycohydrolases II and I were around 7.4 and 7.0, respectively. The Km value for (ADP-ribose)n (average chain length n = 15) and the Vmax for glycohydrolase II were 4.8 microM and 18 mumol of ADP-ribose released from (ADP-ribose)n.min-1.(mg of protein)-1, respectively. The Km was about 2.5 times higher, and Vmax 2 times lower, than those observed with glycohydrolase I. Unlike glycohydrolase I, glycohydrolase II was inhibited by monovalent salts. ADP-ribose and cAMP inhibited glycohydrolase II more strongly than glycohydrolase I. These results suggest that eukaryotic cells contain two distinct forms of poly(ADP-ribose) glycohydrolase exhibiting differences in properties and subcellular localization.     

Strategy for selection of cell variants deficient in poly(ADP-ribose) polymerase

A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.