A Critical Review of PermeabilA critical review of permeabilized cell systems for studying mammalianized Cell Systems for Studying Mammalian

Mutation Research, 236 (1990) 239-252 DNA Repair Elsevier

MUTDNA 06008

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A critical review of permeabilized cell systems for studying mammalian DNA repair

Scott Keeney and Stuart Linn Dioision of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 (U.S.A.)

(Accepted 12 March 1990)

Keywords: Permeabilized cell systems; DNA repair, mammalian; ATP, in DNA repair; DNA polymerase enzymology

Summary

Permeabilized cell systems have proven valuable for studies of the enzymology of mammalian DNA repair and this review will summarize and contrast the different systems used to this end. Results from permeable cell studies will be reviewed which pertain to 3 questions of DNA repair: the role(s) of ATP, DNA polymerase enzymology, and the isolation of repair factors by in vitro correction of repair-defective cells.

Mechanisms for responding to genomic damage are extremely complex, and only in prokaryotes are they becoming understood in detail. Studies of the enzymology of DNA repair in mammalian cells have been particularly hampered by the very limited genetic tools available, forcing the devel- opment of a large number of novel biochemical techniques. One of these, permeabilized cells, is the subject of this chapter.

In 1970, Moses and Richardson described one of the first permeable cell systems for studying DNA repair. They used toluene treatment to per- meabilize E. coli to nucleoside triphosphates and

Correspondence: Dr. Stuart Lima, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 (U.S.A.), phone (415) 642-7583; FAX (415) 643-5035.

Research from our laboratory cited in this review was supported by the U.S. Department of Energy (contract 76EV30415) and the U.S.P.H.S. (Grants RO1GM30415 and P30ES01896). S.K. is supported by aU.S.N.S.F, graduate fellowship.

other small molecules and observed both replicative and repair DNA synthesis with properties similar to those observed in vivo. Open cell approaches analogous to this one have subsequently proven to be useful in studies of mammalian cells as well, as such systems are a valuable complement to in vivo experiments. The major strength of permeable cell systems is the fact that the majority of the cellular DNA-repair machinery is left intact by the permeabilization procedure. Thus permeable cells are capable of carrying out all of the steps of DNA repair, from damage-dependent incision to ligation of repair patches and rearrangements of chromatin structure (Dresler et al., 1982; Dresler and Lieberman, 1983b; Seki et al., 1989).

The elimination of permeability barriers allows the routine introduction of non-permeable factors such as charged molecules and polypeptides into the assay system. For this purpose, a number of irreversible and transient permeabilization techniques have been developed. Many of these sys-

0921-8777/90/$03.50 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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tems are absolutely dependent on exogenously supplied substrates and cofactors such as ATP, dNTPs, and Mg 2+. Such requirements permit the use of defined, experimentally manipulatable concentrations of these molecules free from inter- ference from endogenous pools. This ease of manipulation has been especially valuable for studies of nucleotide requirements and inhibitor effects.

Several lines of evidence indicate that the repair processes observed in open cells are biologically relevant. Repair is damage-dependent and is ab- sent in repair-defective cells (Ciarrocchi and Linn, 1978; Castellot et al., 1979; Roberts and Lieber- man, 1979). Parameters such as dose-responses and sensitivity to inhibitors correlate well with in vivo observations (Ciarrocchi and Linn, 1978; Castellot et al., 1979; Ciarrocchi et al., 1979; Dresler and Lieberman, 1983b). Also, DNA repair synthesis in permeable cells is conservative, with repair patch sizes similar to those measured in vivo (Ciarrocchi and Linn, 1978; Smith and Hanawalt, 1978; Roberts and Lieberman, 1979). Furthermore, cells are viable after repair in certain transient permeabilization systems (Tanaka et al., 1977; Lorenz et al., 1988). Parenthetically, many of the cell permeabilization techniques used to study DNA repair can be adapted to support semi-conservative replicative DNA synthesis in exponential cell populations (Ciarrocchi and Linn, 1978; Berger et al., 1979; Castellot et al., 1979; Hanaoka et al., 1979).

The main disadvantage of permeable cell approaches lies in the potential for artifactual responses under nonbiological reaction conditions. Also, differences in the manner of permeabilization may perturb some systems, making detailed comparisons between different systems difficult. Careful design and optimization of experimental conditions should obviate most of these difficulties which are, after all, the same sorts of problems encountered with any in vitro approach.

A wealth of permeable cell studies of DNA repair (and replication) has accumulated during the past 15 years. Since no summary of these exists, a review of them is timely. This review will stress studies related to 3 areas of mammalian DNA repair: the roles of ATP, the DNA polymerase enzymology of repair, and the identifica-

tion of cellular repair factors through permeable cell complementation studies.

Early permeable cell systems

The first permeable cell system developed for studies of DNA repair in mammalian cells used Sendai virus treatment to introduce a heterologous repair enzyme into xeroderma pigmentosum cells (Tanaka et al., 1975). Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a clinical and cellular hypersensitivity to UV light (reviewed by Friedberg, 1985). Cells from XP patients are deficient in the excision step in DNA repair induced by UV-irradiation or several chemical mutagens. Using Sendai virus-mediated permeabilization, Tanaka and coworkers (1975, 1977) demonstrated that the cyclobutane dimer-specific endonuclease of phage T4 could restore UV-induced unscheduled DNA synthesis in XP cells. This study was significant not only because it demonstrated that XP cells are fully capable of carrying out post-incision repair events, but also because it set the stage for a host of further studies of DNA repair in permeabilized cells.

Several different techniques for permeabilizing cells were developed in subsequent years. Many of these were originally defined to study replicative DNA synthesis, and subsequently adapted to DNA-repair studies. These permeabilization techniques fall into 3 general categories: (i) treatment with detergents such as Brij-58 (Reinhard et al., 1977), lysolecithin (Miller et al., 1978), or saponin (Kaufmann and Briley, 1987); (ii) hypotonic lysis (Ciarrocchi and Linn, 1978; Smith and Hanawalt, 1978; Berger et al., 1979; Hanaoka et al., 1979); (iii) mechanical disruption (Roberts and Lieber- man, 1979). The various techniques also differ from one another at several steps in the procedure. For example, permeabilization can be carried out on cells attached to culture dishes (Castellot et al., 1979; Kaufmann and Briley, 1987) or in suspen- sion (Ciarrocchi and Linn, 1978; Roberts and Lieberman, 1979). Also, the ionic strength of the permeabilization medium varies from system to system. This parameter is particularly important for generating repair- and replication-competent permeable cells as illustrated by the fact that permeabilization by detergent in high salt con-

centrations depletes fibroblasts of DNA polymerase c required for repair synthesis (see below).

In contrast to the differences between various permeabilization techniques, the conditions under which the repair process itself is studied are fairly similar from system to system. For example, repair DNA synthesis in all of the systems cited above depends on added dNTPs and ATP. Fur- thermore, the general requirements for repair DNA synthesis in open cells are distinct from those for replicative synthesis in similar permeable systems. Thus, for example, UV-repair DNA synthesis is inhibited by salt (Roberts and Lieberman, 1979) while replicative DNA synthesis is stimulated by NaC1 (Ciarrocchi et al., 1979; Castellot et al., 1979). Also, repair and replication can be differen- tiated in that replicative synthesis is optimal in permeabilized cells from logarithmic cultures, while UV-repair synthesis is optimal in permeabilized G O cells (Ciarrocchi and Linn, 1978).

Several different events in DNA-repair path- ways have been studied with permeable cells. The most commonly studied are repair DNA synthesis and incision, but such processes as repair patch ligation (Seki et al., 1989) and assembly of repair patches into nucleosomes (Dresler et al., 1982) have also been examined.

A pattern was established early on of using open-cell systems to pursue three basic lines of research: (a) the requirements of DNA-repair for such molecules as ATP, Mg 2, and dNTPs; (b) the effects of inhibitory substances on DNA-repair; and (c) the identification of repair factors by in vitro complementation (patterned after the pro- totypical T4 endonuclease V experiments). This early work provided the impetus for the many detailed studies that followed.

Assessing metabolic requirements: the role of ATP

Essentially every open-cell system that is proficient in carrying out DNA repair in response to UV-irradiation shows a requirement for or stimulation by ATP (see, for example, Smith and Hanawalt, 1978; Roberts and Lieberman, 1979; Dresler and Lieberman, 1983b). DNA repair induced by bleomycin has also been shown to require ATP (Seki et al., 1989). Systems which do not exhibit an absolute dependence upon exoge-

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nous ATP include those in which soluble cellular material is diluted but not removed (e.g. Ciarroc- chi and Linn, 1978) or those which measure repair synthesis after lengthy post-damage incubations prior to permeabilization (Hanaoka et al., 1979), during which time ATP-dependent repair processes may occur.

By measuring the accumulation of strand breaks in permeable human fibroblasts in the presence or absence of ATP, it was demonstrated that at least one ATP-requiring step in the UV-induced excision-repair pathway occurs before or during incision (Dresler and Lieberman, 1983b). Further- more, incubation of UV-irradiated permeable cells in the absence of dNTPs results in the accumulation of DNA breaks in normal cells only if ATP is present; as expected, this phenomenon is not observed in cells from XP patients (Dresler and Lieberman, 1983b; Kaufmann and Briley, 1987). In bleomycin-treated permeable cells, repair DNA synthesis requires ATP, but it is not clear whether this requirement occurs before or during polymerization (Seki et al., 1989).

It is not known where in the pathway leading up to incision ATP is required. For the UvrABC 'excinuclease' of E. coli, damage recognition, nucleoprotein complex assembly, strand sep- aration, and strand scission each require either the binding or hydrolysis of ATP or, in some cases, other triphosphates (Caron and Grossman, 1988). Analogous requirements may operate in mammalian cells in one or all of these cases.

In mammalian cells, other processes might also require ATP. Nucleosomal rearrangements, for example, might be required prior to incision (Smer- don and Lieberman, 1978), either to move nucleosomes or to modify chromatin proteins. In- terestingly, the RAD6 protein of S. cereoisiae has been shown to ubiquitinate histones in vitro (Jentsch et al., 1987). Ubiquitin transfer is an ATP-dependent process (reviewed in Hershko, 1988), and RAD6-mediated ubiquitination could play a role in DNA repair.

Large, ATP-dependent topological changes in DNA may also be required prior to incision. For example, novobiocin inhibits both incision and repair DNA synthesis in UV-irradiated and MNNG-treated cells, both in vivo and in permeable cells (Collins and Johnson, 1979; Mattern

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and Scudiero, 1981; Dresler and Robinson-Hill, 1987). Novobiocin inhibits mammalian DNA polymerase a (Dresler and Robinson-Hill, 1987) and type 2 topoisomerases (Miller et al., 1981), many of which require ATP (Cozzarelli, 1980). The inhibition of repair synthesis could be attri- buted to polymerase inhibition, but the decrease in incision may be due to inhibition of the topoisomerase. This idea was challenged by studies of Downes et al. (1985) who demonstrated gross al- terations in mitochondrial structure along with a decrease in the ATP :ADP ratio in novobiocin- treated cells. These observations suggested that the effect of novobiocin on repair might be the result of ATP-depletion rather than a direct result of inhibition of topoisomerase activity. Other studies (Dresler and Robinson-Hill, 1987) have shown, however, that novobiocin has a direct effect on UV-induced incision in permeable human cells in which repair is absolutely dependent upon exogenous ATP and is presumably insensitive to changes in mitochondrial metabolism. It thus appears that the action of a topoisomerase (or some other novobiocin-sensitive, ATP-dependent activity) is required prior to damage-dependent incision in mammalian cells.

In addition to pre-incision events, ATP appears to play a role in post-incision repair processes. Repair DNA synthesis itself does not require ATP in permeable human fibroblasts, although the data do not rule out a stimulatory role (Dresler and Lieberman, 1983b). Of course, ATP is a cofactor for ligation, but possible ATP requirements for post-ligation events such as chromatin rearrange- ment (Smerdon and Lieberman, 1978) have not been tested.

It is clear from the above observations utilizing permeabilized cells that ATP is an essential par- ticipant at several stages in DNA repair. More detailed characterization of its roles awaits biochemical studies of the various ATP-dependent steps. In particular, it remains to be determined which steps require hydrolysis of ATP as opposed to binding alone and whether other rNTPs can substitute for ATP at any of these steps, as is the case in toluene-permeabilized E. coli (Masker and Hanawalt, 1974). This last question is particularly interesting in light of the ability of GTP to substitute for ATP in the NTPase activity of UvrA (Caron and Grossman, 1988).

DNA polymerases

A key component of the process of DNA excision repair is the polymerase responsible for re- synthesis of the excised DNA. Mammalian cells contain 5 known DNA polymerases, ct, /3, y, 8 and c (reviewed in Fry and Loeb, 1986; Burgers, 1989; Burgers et al., 1990). Polymerase a is thought to act in DNA replication, and there is evidence for a role in DNA repair as well. Polymerase /3 has been implicated in some forms of DNA repair. Polymerase "t seems to be present in both mitochondria and nuclei. While the mitochondrial fraction is thought to be responsible for replication of the mitochondrial genome, no role has as yet been determined for the nuclear fraction. Poly- merases 8 and ~ (referred to as 81 and 82, respec- tively, in Burgers (1989) or simply as 'polymerase 8' in much of the early literature) have intrinsic 3 ' -5 ' exonuclease activities which have been shown to serve a proofreading function. Polymerase 8 has both its processivity and activity increased dramatically by proliferating cell nuclear antigen (PCNA) and is thought to play a role in DNA replication. Polymerase c, in contrast, is inherently highly processive and is not stimulated by PCNA. Polymerase e has been directly implicated in UV- induced DNA repair by permeable cell complementation studies (see below).

In this section, efforts to use permeable cell systems to elucidate the roles of various polymerases in excision repair processes will be reviewed. Kinetic studies of DNA polymerases in DNA-repair synthesis in permeable cells will also be summarized.

Inhibitor studies One approach to determining which poly-

merase(s) is involved in DNA repair has been the study of repair synthesis in the presence of selective inhibitors of the various known polymerases. Early work of this type in mammalian cells has been extensively reviewed (Fry and Loeb, 1986), but more recent discoveries relating to the roles of DNA polymerases 8 and c warrant a reevaluation of conclusions from these earlier studies.

The use of enzyme inhibitors to study DNA-repair synthesis can be a somewhat problematic

approach to a very complex problem. Difficulties in data interpretation often arise because of the complex kinetics of DNA polymerization. Fur- thermore, degrees of inhibition in vivo or in open cells do not always correlate with observations with purified enzymes. Moreover, the dose and type of damage sustained determine at least in part which polymerase mediates the repair synthesis. For these reasons, the question of whether a given polymerase can be involved might be clearly answered, but more complex questions of interactions or interchangeability among polymerases are not effectively addressed. Despite these problems, however, DNA polymerase inhibitors have provided insight into key facets of the enzymology of DNA repair synthesis. As noted below, the pre- ponderance of published data supports a model in which polymerase c and polymerase /3 are the major repair DNA polymerases, while the roles of polymerase a and polymerase 8 are not clear.

There have been a number of reports of the effects of putatively specific inhibitors of polymerases a and fl in a wide range of systems, both in vivo and in permeabilized cells. Many of these studies revolved around the differential effects of aphidicolin and dideoxyTTP on these enzymes. (Earlier studies did not consider polymerase 8, while later studies probably confused polymerases 8 and c). Aphidicolin, arabinofuranosyl nucleotide analogs (e.g. ara-CTP), and N-ethylmaleimide (NEM) effectively inhibit DNA polymerases et, 8 and c but affect polymerase fl only weakly. Dide- oxyTTP, in contrast, inhibits polymerase fl much more strongly than polymerases a, 8 or c (reviewed in Fry and Loeb, 1986). With these inhibitors, effects upon DNA-repair synthesis are dependent on the DNA-damaging agent used. For example, repair DNA synthesis in human fibroblasts in response to bleomycin and neocarzino- statin is strongly inhibited by ddTTP, with 50% inhibition at 8-16 /xM ddTrP; conversely, 700- 1100 #M ddTTP is required for inhibition of MNNG- or UV-induced repair synthesis in the same system (Miller and Chinault, 1982a, b). Fur- thermore, bleomycin-induced repair synthesis is relatively insensitive to aphidicolin, NEM, ara- CTP and other inhibitors of polymerases a, 8 and c (Castellot et al., 1979; Miller and Chinault, 1982a, b; Dresler et al., 1988). These results are

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consistent with polymerase fl functioning in repair synthesis induced by bleomycin.

Aphidicolin, on the other hand, strongly inhibits repair synthesis in response to UV, MNU or NA-AAF (Berger et al., 1979; Ciarrocchi et al., 1979; Hanaoka et al., 1979; Miller and Chinault, 1982b; Dresler and Lieberman, 1983a), with 80- 90% inhibition of repair at saturating levels of inhibitor. Since polymerase a was the only polymerase known at the time with aphidicolin sensitivity similar to that of repair synthesis in these permeable cells, these results were interpreted as demonstrating a role for polymerase a in excision repair of damage induced by bulky adducts, though, as discussed below, this conclusion ought to be modified.

The above results are consistent with a model in which at least two DNA-repair synthesis path- ways exist in the cell (Miller and Chinault, 1982b), one mediated by polymerase fl and another by an aphidicolin-sensitive DNA polymerase. The nature of the DNA lesion would determine which polymerase mediates repair synthesis, this choice perhaps resting on the nature of the gap or strand break to be resynthesized. For example, bleomycin produces DNA-strand breaks by direct attack on the sugar-phosphate backbone, leaving a 3-carbon fragment from deoxyribose at the 3' terminus (Burger et al., 1980). These termini do not serve as primers for DNA synthesis, but they may be removed by an exonuclease associated with polymerase fl to generate appropriate primers (Seki and Oda, 1988). An analogous system has been demonstrated in vitro, where DNAase V (a nuclease found in association with polymerase fl (Mosbaugh and Linn, 1983)) catalyzes the removal of abasic 3' sugar residues from UV-irradiated DNA treated with T4 endonuclease V and human AP endonuclease II, allowing polymerase fl to carry out gap-filfing synthesis (Mosbaugh and Linn, 1983). Such a system would account for the apparent major role of polymerase fl in bleomycin-induced repair. A role for polymerase 13 in simple base-excision repair is also thought to be likely, but at this time direct evidence is lacking.

In contrast, damaged nucleotide excision might be expected to generate single-strand breaks or gaps with 3' termini capable of supporting DNA synthesis directly. Such breaks may be prefer-

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entially filled in by polymerase a (or 8 or c), explaining the aphidicolin-sensitivity of repair synthesis induced by UV, MNNG or NA-AAF.

The conclusion that DNA polymerase a is responsible for the aphidicolin-sensitive repair seen in UV-damaged cells has been convincingly challenged by several fines of evidence implicating polymerase ~ instead. As mentioned above, polymerases ~ and c are also sensitive to aphidicolin, NEM and ara-CTP, so the earlier studies using these inhibitors are consistent with a role for any of these 3 polymerases in repair of damage induced by UV, NA-AAF and alkylating agents such as MNNG and MNU (Dresler, 1984). How- ever, more recent studies using butyl-phenyl dGTP and ddTTP are more consistent with either polymerase 8 or polymerase c as the major UV-repair polymerase. Specifically, BuPdGTP, a much more potent inhibitor of polymerase ct than of polymerases 8 or c (Burgers, 1989) inhibits UV-induced repair synthesis in permeable human fibroblasts much less strongly than it inhibits polymerase a (Dresler and Frattini, 1986, 1988). Also, UV-repair synthesis is more sensitive than polymerase a to inhibition by ddTTP (Dresler and Kimbro, 1987), to which polymerase c has a sensitivity intermediate between that of polymerases a and fl (Wahl et al., 1986; Syvaoja et al., 1990). Finally, further data from permeable cell complementation studies (Nishida et al., 1988b: described in more detail below) provide direct evidence for the involvement of polymerase c in UV-repair synthesis. It seems likely, then, that either polymerase c or polymerase fl is capable of mediating repair synthesis, depending on the nature of the damage. A role for polymerases a or 8 in some DNA-repair synthesis is not ruled out by these studies, however.

An additional factor in determining which polymerase mediates repair synthesis was suggested by studies of Dresler and colleagues, who reported that the involvement of aphidicolin-sensitive polymerases in repair synthesis is dependent on the damage dose. They demonstrated that repair synthesis in permeable human fibroblasts at low doses of MNU, bleomycin, NA-AAF or UV is more resistant to aphidicolin than is repair synthesis at higher doses (Dresler and Lieberman, 1983a). They concluded that polymerase fl may carry out

a larger percentage of repair synthesis when the extent of damage is low, but that higher doses result in the involvement of aphidicolin-sensitive polymerase(s). In vivo data from UV-damaged cells confirms these observations (Smith and Okumoto, 1984).

However, these conclusions are not supported by data of Keyse and Tyrrell (1985) who showed that, even at low doses of UV, the fraction of repair synthesis resistant to aphidicolin is also resistant to ddTI'P, arguing against involvement of polymerase /3. In fact, a simple kinetic argu- ment can explain these observations without in- voking polymerase ft. At low damage doses, the availability of incision sites would be limiting, allowing the continued presence of a pool of repair-competent, aphidicolin-sensitive polymerase molecules. At higher (saturating) doses, the availability of polymerase molecules becomes limiting. When a polymerase molecule is inhibited by aphidicolin in the process of chain elongation at low damage doses, elongation can resume either by dissociation of the inhibitor or by dissociation of the polymerase from the template, with another polymerase molecule being available to continue synthesis. In contrast, at high damage doses, only dissociation of the inhibitor would result in con- tinuation of DNA synthesis, since no free polymerase molecules would be available. Such a mechanism would account for the decreased sensitivity of the repair system to aphidicolin at low damage doses. This point highlights the caveats laid out at the beginning of this section. Namely, polymerase inhibition is not a simple on/o f f system independent of experimental conditions, since substrate concentration (damaged DNA template and dNTPs) as well as available enzyme amounts may affect the degree of inhibition.

A further example also illustrates this point. While studies generally agree that ddTTP is a weak inhibitor of UV-repair synthesis in permeable cells, they are not consistent on the extent and ddTTP concentration-dependence of the inhibition. Ciarrocchi et al. (1979) reported concentration-dependent inhibition by ddTTP up to 0.8 mM, with maximal inhibition of 80%, while Dresler and Lieberman (1983a) demonstrated maximal inhibition of only 20% reached at 40/~M ddTTP, with higher ddTTP concentrations having

no further effect. Further examination of the data reveals that the differences arose from the different dTTP concentrations used in the assays (0.4 /~M in the former, 3 /xM in the latter). Since ddTTP is competitive with dTTP in polymerization and repair DNA synthesis has a relatively low apparent K m for dNTPs (Dresler, 1984; Dresler et al., 1988) and a relatively high apparent K i for ddTTP (Dresler and Kimbro, 1987), studies using (saturating) levels of dTTP of several micromolar demonstrate little or no inhibition by ddTTP. This may seem at first to be a minor point of enzyme- inhibitor kinetics, but it bears emphasizing here. Many studies treat competitive inhibitors as species which absolutely inhibit the enzyme of interest at a specific concentration regardless of other factors. Failure to interpret inhibitor data with an eye toward the kinetic properties of the system can easily result in naive or incorrect conclusions.

Kinetic studies In addition to the numerous inhibitor studies of

DNA repair, several attempts have been made to measure kinetic properties of repair synthesis (Castellot et al., 1979; Dresler, 1984; Dresler et al., 1988). These studies exploit the fact that DNA repair synthesis in permeable cells is dependent upon exogenously supplied dNTPs, allowing for defined substrate concentrations. This approach requires two main assumptions (Castellot et al., 1979). First, the supplied substrates must have free access to the synthetic apparatus. In terms of the permeability barriers normally posed by cell membranes, this is probably a safe assumption in the systems used in these studies. Second, intracellular dNTP pools must be insignificant or known. It is likely that such pools are negligible compared to exogenous supplies after the washing or dilution steps of most open cell assay systems.

It should also be noted that the Km'S measured are apparent Km'S (K~), which are dependent on reaction parameters other than dNTP concentrations. In particular, primer-template concentration is important in determining polymerase kinetic parameters. In the studies discussed here, this concentration varies depending on the type and dose of damage administered. The effects of other substances (e.g. cofactors such as Mg 2+, inhibitory

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products such as pyrophosphate, and accessory proteins such as PCNA) are also potentially significant. These considerations should be borne in mind when making comparisons between different permeable cell systems or between reactions in open cells and reactions catalyzed by purified enzymes. Furthermore, the extreme complexity of polymerase kinetics is well-documented (see, for example, Detera et al., 1981), making straightfor- ward measurement of kinetic parameters difficult. For example, reaction conditions such as the concentration of Mg 2 affect the processivity of nucleotide incorporation (Syvaoja and Linn, 1989), which in turn affects the nature of the steady-state reaction mechanism (Detera et al., 1981). Even the method used to experimentally vary the dNTP concentrations affects the values of observed kinetic parameters. Specifically, widely different K m s are measured in the same system depending on whether all 4 dNTPs are varied simultaneously or whether one is varied while the other 3 are held constant at saturating levels (see below).

Using lysolecithin-permeabilized BHK cells and bleomycin for a damaging agent, Castellot et al. (1979) measured apparent Km'S of 50 ___ 20/xM for dNTPs in replication and 170 _ 50 /xM in repair. Similarly, studies in mechanically disrupted human fibroblasts (Dresler, 1984) demonstrated an apparent K m of 30 /xM for replicative synthesis, while the apparent K m for UV-induced repair synthesis was 0.07/~M. This large difference between repair values could be due to the involvement of different polymerases, namely polymerase fl in bleomycin-induced repair synthesis and an aphidicolin-sensitive polymerase in UV-induced repair (see above). The difference is unlikely to be affected greatly by the permeabilization technique, since UV-induced repair synthesis in human fibroblasts permeabilized with lysolecithin was also reported to have an apparent K m of 0.1 /~M (Lorenz et al., 1988).

The above apparent Km'S were determined by varying the concentrations of all 4 dNTPs simultaneously over a range of values. Further studies demonstrated very different kinetics for repair and replicative synthesis in the same permeable cell system when 3 dNTPs were held constant at saturating levels and the fourth was varied. Thus, apparent Km'S of 1.2-2.9 /xM (depending on the

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cell type and dNTP varied) were measured for replicative synthesis and 0.06-0.44 ~M for UV-induced repair synthesis in permeable human fibroblasts (Dresler, 1984; Dresler et al., 1988). The reason for the differences between these values and the previous ones is not clear. They are not artifacts of the permeable cell system, however, since purified polymerase from HeLa cells dem- onstrates similar variations in vitro (J. Syvaoja and S. Linn, unpublished observations). Instead, the kinetic complexity of DNA polymerization in general probably underlies these results.

These complexities aside, there remains the observation that apparent gm'S for UV-induced repair synthesis are vastly lower than those for replication measured under the same conditions. These differences, like the ones discussed above, do not appear to have an easily definable cause, but there are some important considerations. First, both replicative and repair synthesis are studied in the absence of rNTPs, which have been shown to stimulate replicative synthesis in some permeable cell systems (Castellot et al., 1979), perhaps by serving as precursors for primer formation. If replication fork models (Downey et al., 1988; Prelich and Stillman, 1988) which postulate coordinated leading and lagging strand synthesis in mammalian cells are correct, the inability to form lagging strand primers could have an effect on the rate of synthesis. Second, the template-primer concentrations in replicative and repair synthesis are likely to be quite different, perhaps severely altering observed gin's for the two processes. Third, it has been reported that thymidine is preferred over dTTP as a precursor for DNA synthesis in osmotically disrupted fibroblasts, while dT'FP is preferred for repair synthesis in the same system (Ciarrocchi and Linn, 1978). Thus, differences in apparent Km'S for repair and replication may simply reflect a differential facility to use exogenous dNTPs as precursors for synthesis. Finally, current models suggest that different polymerases mediate replication and UV-repair, with polymerases a and 8 carrying out replication (Burgers, 1989) and polymerase c mediating the majority of UV-induced repair synthesis (discussed in this review). If this is the case, the observation of different kinetics for events using different enzymes is not surprising.

Despite these considerations, it is certainly possible that these observed differences are real, in which case there are several interesting implications. For example, there may be an advantage to having a low K m for dNTPs in repair synthesis since responses to damage must often occur in quiescent cells with low dNTP levels (Dresler, 1984; Snyder, 1984). These lower Km's may also contribute to the relative resistance of repair synthesis to inhibition by hydroxynrea (Dresler, 1984), which significantly reduces intracellular dNTP pools by inhibiting ribonucleotide re- ductase (Snyder, 1984). It has also been pointed out (Dresler et al., 1988) that, while the apparent g m for dNTPs in repair is lower than in replication, there is little difference in the Ki's measured for inhibition of these processes by BuPdGTP and ddTTP. Lower Km/K i ratios may be indicative of the ability of the repair apparatus to more effectively discriminate against altered substrate nucleotides. This is especially significant in light of the fact that free dNTP pools are targets of many DNA-damaging agents (Topal, 1985). All of these possibilities add up to a consid- erable potential selective advantage for low gm's for dNTPs in repair DNA synthesis.

An additional facet of these kinetic studies is the utilization of correlations and/or differences between apparent Km'S measured in permeable cells and those with purified enzymes to assign DNA synthesis seen under various conditions to specific polymerases. Thus, for example, the above apparent Km'S for replication are consistent with published values for polymerase ct (Detera et al., 1981). Such arguments are not very convincing, however, since large discrepancies are seen in some cases. Compare, for example, the apparent K m of less than 0.4/~M for repair synthesis in UV-treated cells with the apparent g m of ca. 2-5 ttM reported for polymerase ~ isolated from calf thymus or HeLa cells (Dresler et al., 1988; Syvaoja and Linn, 1989). Dresler and coworkers suggested (1988) that these differences may be due to association of the polymerase in vivo with accessory proteins which alter the enzyme's kinetic properties. There is precedence for this phenomenon in, for example, the phage T4 replicative system: the T4 polymerase by itself has a forty-fold lower K m for dNTPs than when assembled into a replicative

complex (Mathews and Sinha, 1982). However, there are other possible explanations as well. For example, as mentioned earlier, the concentration of template-primer in permeable cells differs from that in vitro, affecting the value of the apparent Km. In addition, assigning specific roles to the various polymerases based on kinetic data as- sumes that only one polymerase is responsible for the synthesis measured. This assumption is dubi- ous in the cases of both replicative and repair DNA synthesis. Specifically, the inhibitor studies discussed above implicate both polymerase fl and an aphidicolin-sensitive polymerase in repair of bleomycin-induced damage (Castellot et al., 1979; Miller and Chinault, 1982a, b), while there is good evidence that both polymerase a and polymerase 8 function in replication (reviewed in Burgers, 1989).

In conclusion, it appears that detailed polymerase kinetic studies have yielded results that are provocative, but not definitive. Furthermore, it is important to keep in mind when carrying out such kinetic analyses that the kinetics themselves are quite complex, potentially subject to a wide range of complications in roughly defined systems.

Isolating repair factors

One of the principle goals in mammalian DNA repair research is to identify factors responsible for carrying out a repair process. A number of different approaches are being employed to achieve this goal. These include molecular cloning approaches (reviewed in Friedberg et al., 1987); purification of enzymatic activities presumed to function in repair, such as DNA-glycosylases, AP endonucleases (and lyases), and damage-binding proteins (see, for example, Kim and Linn, 1988; Chu and Chang, 1988; O'Connor and Laval, 1989); and purification of repair factors by complementation of repair-defective cells through microinjection (de Jonge et al., 1983) or permeabilization (Nishida et al., 1988b). Some of these approaches are reviewed elsewhere in this volume. We will focus on the use of permeable cells in in vitro complementation experiments.

Polymerase epsilon as a repair factor Reinhard et al. (1979), working with CHO cells

and the non-ionic detergent Brij-58, reported that

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cells permeabilized and depleted of soluble com- ponents in the presence of high concentrations of detergent and KC1 were incapable of carrying out replicative or UV-repair DNA synthesis. UV-dependent repair synthesis could be restored, however, by addition of whole cell extracts from repair-proficient cells. A similar system using normal human fibroblasts served as the basis for the discovery of a soluble factor from HeLa cells which restored repair synthesis to permeabilized cells.

Extensive purification of this factor from HeLa cells yielded an activity associated with a 220-kD polypeptide with the DNA polymerase and 3 ' -5 ' exonuclease activities reported by Crute et al. (1986) for DNA polymerase 811 (Nishida et al., 1988b). This purified enzyme was not recognized by monoclonal antibodies directed against polymerase a, nor was it as sensitive as polymerase ot to inhibition by BuPdGTP and BuAdATP (Nishi- da et al., 1988b), in agreement with the character- istics of polymerase 811 (referred to here as polymerase c) from calf thymus (Wahl et al., 1986). This was the first direct demonstration of a role for a delta-like polymerase in DNA repair, con- firming the results of inhibitor studies discussed above. It is important to note, however, that though neither purified polymerases a or fl can substitute for polymerase c in this permeable cell system, their participation is not ruled out. These results simply indicate that they are not limiting factors for DNA-repair synthesis under these conditions.

Further characterization of HeLa DNA polymerase c demonstrated that it is not only structurally and immunologically distinct from polymerase a (Wong et al., 1989), but that in spite of its 3 ' -5 ' exonuclease, it differs from purified DNA polymerase 8 preparations as well (see Burgers, 1989). In particular, the repair polymerase is highly processive and is not stimulated by PCNA (Syvaoja and Linn, 1989).

A number of lines of evidence suggest that DNA polymerases 8 and ~ fulfill different roles in the cell. The lack of stimulation of polymerase c by PCNA is particularly interesting. PCNA is a cell cycle-regulated protein which has been implicated in DNA replication because it is required for efficient SV40 DNA replication in vitro (Pre-

248

lich et al., 1987a, b; Prelich and Stillman, 1988). It stimulates both the activity and the processivity of polymerase 6 on templates containing long, single-stranded regions (Tan et al., 1986). These observations suggest that polymerase ~ may be an important replicative polymerase. One model pos- tulates that polymerase ~ is the leading strand polymerase in a multi-protein complex with PCNA, perhaps with polymerase a-primase serving as the lagging strand polymerase (Downey et al., 1988; Prelich and Stillman, 1988). Whatever the role of polymerase 8 in replication, however, it seems that polymerases 6 arid c serve very different roles in the cell.

We have recently isolated, in addition to polymerase c, a PCNA-sensitive polymerase 6 from HeLa cells, giving us an opportunity for the first time to compare these two enzymes from the same cell type (Syvaoja et al., 1990). Polymerase ~ has two subunits with Mr's of 134 and 47 kDa, whereas polymerase c has two subunits with Mr'S of 215 and 55 kDa. HeLa polymerase ~, but not HeLa polymerase ~ has UV-repair activity in the permeable cell assay.

It is interesting to note that there are two (~elta-like DNA polymerases in S. cereoisiae as ,Nell (Burgers, 1989). The yeast enzymes, DNA polymerases II and III, both contain intrinsic 3 ' -5 ' exonuclease activities. Polymerase II I appears to be related to mammalian polymerase 6 since it is stimulated by PCNA purified from yeast or calf thymus (Bauer and Burgers, 1988). Moreover, yeast polymerase III is implicated in DNA replication since temperature-sensitive mutations of the CDC2 gene which codes for the enzyme (Sitney et al., 1989; Boulet et al., 1989) show defective DNA synthesis at the restrictive temperature (Pringle and Hartwell, 1981). In contrast, the subunit structure and PCNA-insensitivity of yeast DNA polymerase II suggest that it is related to mammalian DNA polymerase ~ (Hamatake et al., 1989). The in vivo role of DNA polymerase II is currently unknown: it will be very interesting to see if it is involved in DNA repair.

Further studies on the role of polymerase c in DNA repair are also indicated. The inhibitor studies discussed above strongly suggest that it is responsible for a majority of the UV-induced DNA-repair synthesis in human fibroblasts, but

questions remain about specific details. Does the 3' 5' exonuclease play a role in addition to its proofreading function? It might, for example, re- move damaged 3' termini from a primer-template, analogous to the activity of human DNAase V in concert with polymerase/3 (Mosbaugh and Linn, 1983). It is also not known whether polymerase has a role in stimulating incision directly, perhaps by interacting with the incision machinery.

Identification of XP correcting factors Although purified polymerase ~ alone restores

UV-induced repair synthesis to permeabilized normal human fibroblasts, it fails to support such repair in XP group A cells (Nishida et al., 1988b). Supplying the permeable XP-A cells with T4 endonuclease V in addition to polymerase ~, however, results in stimulation of high levels of DNA- repair synthesis (Nishida et al. 1988a, b). This observation suggested an in vitro complementation system for purifying cellular factors which correct XP excision defects. Thus, polymerase plus a whole-cell or fractionated extract from repair-proficient cells is capable of stimulating repair synthesis in permeable XP fibroblasts of groups A, C, D, E and I (Nishida et al., 1988a and unpublished). Specifically, extracts of HeLa cells effectively correct XP-A, C, E and I cells, while extracts of normal human fibroblasts and the murine MPC-11 cell line stimulate UV-induced repair synthesis in XP-C and XP-D cells.

We have established 3 basic criteria for identifying XP correcting factors using this system: (a) the purified factor must correct the defect in cells of an XP group; (b) the purified factor must not correct the defect in other XP groups; (c) the factor must be missing or altered in cells of the group it corrects.

The chromatographic properties of DNA polymerase ~ and the XP-A, C, D, and E correcting factors are distinct (Table 1), suggesting that they are separate entities. Table 2 summarizes the results of cross-complementation studies using partially purified XP correcting factors. None of the factors corrects the defect in more than one XP group. (The XP-A correcting factor copurifies with the XP-E factor through phosphocellulose chro- matography (Table 1), but further purification of the XP-E factor through DEAE-cellulose and

TABLE 1

THE XP-A, XP-C, XP-D AND XP-E CORRECTING FAC- TORS AND DNA POLYMERASE HAVE DISTINCT CHROMATOGRAPHIC PROPERTIES

Correcting Source factor

[Salt] required for elution

Phosphocellulose DEAE

XP-A HeLa 210 mM KP i (pH 7.5)

XP-C human fibro- 350 mM KCI blasts and murine MPC-11

XP-D human fibro- PT blasts

XP-E HeLa 220 mM KP i (pH 7.5)

DNA po lc HeLa 290 mM NaC1

ND

PT

125 mM KP i (pH 8.1)

175 mM NaC1 (pH 7.5)

220 mM NaC1

Soluble extracts were prepared from the indicated sources, chromatographed, and assayed for correcting factor activity as described in Nishida et al. (1988a, b). Abbreviations: ND, not determined; PT, pass-through fraction; KPi, potassium phosphate.

heparin agarose columns results in loss of the ability to correct XP-A cells, suggesting that they are separate activities.)

TABLE 2

EFFECT OF PARTIALLY PURIF IED XP CORRECTING FACTORS ON VARIOUS COMPLEMENTATION GROUPS

Permeabilized fibroblasts

XP correcting factor a

XP-A XP-C XP-D XP-E

GM2990 (XP-A) + _ b _ - -

GM0709 (XP-C) ND + c ND -

GM5424 (XP-D) - _ b + _

GM2415 (XP-E) (+) d -- b -- +

a Partially purified XP correcting factors were assayed in the permeable cell assay as described in Nishida et al. (1988a, b) using the indicated XP cell strains. Correcting factors were purified from HeLa cells unless otherwise indicated. ND, not determined.

b Murine MPC-11 factor. c Human fibroblast and murine MPC-11 factors. d The phosphocellulose fraction of the XP-A factor contains

XP-E factor activity. See text.

249

Of the XP factors studied in our laboratory, the XP-D factor is the best characterized to date, meeting all 3 of the above criteria. It has been shown (Nishida et al., 1988a and unpublished) that the XP-D correcting activity from MPC-11 cells and normal human fibroblasts copurifies with UV endonuclease III. UV endonuclease III activity is missing from XP-D lymphoblasts, which contain normal levels of UV endonucleases I and II (Kim and Linn, 1988). UV endonuclease III has been extensively purified from MPC-11 cells. It is a 3.2S enzyme which nicks heavily UV-irradiated and OsO4-treated DNA. In addition, this enzyme nicks DNA at AP sites and is, in fact, identical to human AP endonuclease I. This observation agrees with earlier data showing that XP-D fibroblasts are deficient in AP endonuclease I (Kuhnlein et al., 1978).

Several important questions about this enzyme remain unanswered at this point. For example, the exact nature of the lesion(s) recognized is not known, although the fact that UV endonuclease III nicks both OsO4-treated and heavily UV- irradiated (500 J /m 2) but not lightly irradiated DNA (23 J /m 2) suggests that it may recognize pyrirnidine hydrates. It is also not yet known whether the enzyme has glycosylase activity as might be expected from the fact that it has AP-endonuclease activity. Interestingly, this enzyme strongly resembles a relatively unstudied enzyme from E. coli, endonuclease V (Demple and Linn, 1982).

Conclusions

A number of permeabilized mammalian cell systems have been developed to study mammalian DNA repair. These systems are extremely useful given the absence of a solid genetic basis for studying mammalian DNA repair. While these systems have obvious advantages over studies of either whole cells or cell-free extracts, they also have some of the disadvantages associated with each, particularly for obtaining definitive interpre- tations of results. Nonetheless, these systems have been used successfully for identifying DNA-repair factors and may be exploited for purification of such factors prior to learning their biochemical basis. Thus, with proper caution, permeabilized

250

cel l sys tems shou ld be use fu l for impor tant s tud ies o f DNA repa i r .

References

Bauer, G.A., and P.M.J. Burgers (1988) The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen inter- acts with mammalian DNA polymerase 5, Proc. Natl. Acad. Sci. (U.S.A.), 85, 7506-7510.

Berger, N.A., K.K. Kurohara, S.J. Petzold and G.W. Sikorski (1979) Aphidicolin inhibits eukaryotic DNA replication and repair - - implications for involvement of DNA polymerase a in both processes, Biochem. Biophys. Res. Com- mun., 89, 218-225.

Boulet, A., M. Simon, G. Faye, G.A. Bauer and P.M.J. Burgers (1989) Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase IIl, EMBO J., 8, 1849-1854.

Burger, R.M., A.R. Berkowitz, J. Peisach and S.B. Horwitz (1980) Origin of malondialdehyde from DNA degraded by Fe(II)-bleomycin, J. Biol. Chem., 255, 11832-11838.

Burgers, P.M.J. (1989) Eukaryotic DNA polymerases a and 5: Conserved properties and interactions, from yeast to mammalian cells, Prog. Nucleic Acids Res. Mol. Biol., 37, 235- 280.

Burgers, P.M.J., R.A. Bambara, J.L. Campbell, L.M.S. Chang, K.M. Downey, U. Hiibscher, M.Y.W.T. Lee, S.M. Linn, A.G. So and S. Spadari (1990) Revised nomenclature for eukaryotic DNA polymerases, Ear. J. Biochem., in press.

Caron, P.R., and L. Grossman (1988) Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair, Nucleic Acids Res., 16, 9651-9662.

Castellot, J.J., Jr., M.R. Miller, D.M. Lehtomaki and A.B. Pardee (1979) Comparison of DNA replication and repair enzymology using permeabilized baby hamster kidney cells, J. Biol. Chem., 254, 6904-6908.

Chu, G., and E. Chang (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA, Science, 242, 564-567.

Ciarrocchi, G., and S. Linn (1978) A cell-free assay measuring repair DNA synthesis in human fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 75, 1887-1891.

Ciarrocchi, G., J.G. Jose and S. Linn (1979) Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase ct in DNA repair, Nucleic Acids Res., 7, 1205-1219.

Collins, A., and R. Johnson (1979) Novobiocin: an inhibitor of the repair of UV-induced but not X-ray-induced damage in mammalian cells, Nucleic Acids Res., 7, 1311-1320.

Cozzarelli, N.R. (1980) DNA Topoisomerases, Cell, 22, 327- 328.

DeJonge, J.R., W. Vermeulen, B. Klein and H.J. Hoeijmakers (1983) Microinjection of human cell extracts corrects xeroderma pigmentosum defect, EMBO J., 2, 637-641.

Demple, B., and S. Linn (1982) On the recognition and cleav- age mechanism of Escherichia coil endodeoxyribonuclease

V, a possible DNA repair enzyme, J. Biol. Chem., 257, 2848-2855.

Detera, S.D., S.P. Becerra, J.A. Swack and S.H. Wilson (1981) Studies on the mechanism of DNA polymerase a, J. Biol. Chem., 256, 6933-6943.

Downes, C.S., M.J. Ord, A.M. Mullinger, A.R.S. Collins and R.T. Johnson (1985) Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases, Carcinogenesis, 6, 1343-1352.

Downey, K.M., C.-K. Tan, D.M. Andrews, X. Li and A.G. So (1988) Proposed roles for DNA polymerases a and 5 at the replication fork, in: T. Kelly and B. Stillman (Eds.), Cancer Cells, 6. Eukaryotic DNA Replication, Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 403-410.

Dresler, S.L. (1984) Comparative enzymology of ultraviolet-induced DNA repair synthesis and semiconservative DNA replication in permeable diploid human fibroblasts, J. Biol. Chem., 259, 13947-13952.

Dresler, S.L., and M.G. Frattini (1986) DNA rephcation and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase a to inhibition by butylphenyl-deoxyguanosine triphosphate, Nucleic Acids Res., 14, 7093-7102.

Dresler, S.L., and M.G. Frattini (1988) Analysis of butylphenyl-guanine, butylphenyl-deoxyguanosine, and butylphenyl-deoxyguanosine triphosphate inhibition of DNA rephcation and ultraviolet-induced DNA repair synthesis using permeable human fibroblasts, Biochem. Pharmacol., 37, 1033-1037.

Dresler, S.L., and K.S. Kimbro (1987) 2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: Evi- dence for involvement of DNA polymerase 5, Biochem- istry, 26, 2664-2668.

Dresler, S.L., and M.W. Lieberman (1983a) Identification of DNA polymerases involved in DNA excision repair in diploid human fibroblasts, J. Biol. Chem., 258, 9990-9994.

Dresler, S.L., and M.W. Lieberman (1983b) Requirement of ATP for specific incision of ultraviolet-damaged DNA during excision repair in permeable human fibroblasts, J. Biol. Chem., 258, 12269-12273.

Dresler, S.L., and R.M. Robinson-Hill (1987) Direct inhibition of u.v.-induced DNA excision repair in human cells by novobiocin, coumermycin and nalidixic acid, Carcinogene- sis, 8, 813-817.

Dresler, S.L., J.D. Roberts and M.W. Lieberman (1982) Char- acterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts, Biochemistry, 21, 2557-2564.

Dresler, S.L., M.G. Frattini and R.M. Robinson-Hill (1988) In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells, Biochemistry, 27, 7247-7254.

Friedberg, E.C. (1985) DNA Repair, Freeman, San Francisco. Friedberg, E.C., C. Backendorf, J. Burke, A. Collins, L. Gross-

man, J.H.J. Hoeijmakers, A.R. Lehmann, E. Seeberg, G.P. van der Schans and A.A. van Zeeland (1987) Molecular aspects of DNA repair, Mutation Res., 184, 67-86.

Fry, M., and L.A. Loeb (1986) Animal Cell DNA Polymerases, CRC Press, Boca Raton, FL.

Hamatake, R.K., H. Hasegawa, A.B. Clark, K. Bebenek, T.A. Kunkel and A. Sugino (1989) Purification and characterization of DNA polymerase II from the yeast Saccharomyces cereoisiae: Identification of the catalytic core and a possible holoenzyme form of the enzyme, J. Biol. Chem., 265, 4072- 4083.

Hanaoka, F., H. Kato, S. Ikegami, M. Ohashi and M. Yamada (1979) Aphidicolin does inhibit repair replication in HeLa cells, Biochem. Biophys. Res. Commun., 87, 575-580.

Hershko, A. (1988) Ubiquitin-mediated protein degradation, J. Biol. Chem., 263, 15237-15240.

Jentsch, S., J.P. McGrath and A. Varshavsky (1987) The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme, Nature (London), 329, 131-134.

Kaufmarm, W.K., and L.P. Briley (1987) Reparative strand incision in saponin-permeabilized human fibroblasts, Mu- tation Res., 184, 237-243.

Keyse, S.M., and R.M. Tyrrell (1985) Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: Evidence that a- and fl-polymerases act sequentially at the repolymerisation step, Mutation Res., 146, 109-119.

Kim, J., and S. Lima (1989) Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells, J. Biol. Chem., 264, 2739-2745.

Kim, J., S.-Y. Choi and S. Linn (1988) Purification and characterization of three 'UV endonuclease' activities from mammalian cells, J. Cell. Biochem., Suppl. 12A, Liss, New York, p. 268.

Kuhnlein, U., B. Lee, E.E. Penhoet and S. Linn (1978) Xero- derma pigmentosum fibroblasts of the D group lack an apurinic DNA endonuclease species with a low apparent Kin, Nucleic Acids Res., 5, 951-960.

Lorenz, J.D., J.F. Watkins and M.J. Smerdon (1988) Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin, Mutation Res., 193, 167-179.

Masker, W.E., and P.C. Hanawalt (1974) Nucleoside triphosphate dependence of repair replication in toluenized Escherichia coli, J. Mol. Biol., 88, 13-23.

Mathews, C.K., and N.K. Sinha (1982) Are DNA precursors concentrated at replication sites? Proc. Natl. Acad. Sci. (U.S.A.), 79, 302-306.

Mattern, M.R., and-D.A. Scudiero (1981) Dependence of mammalian DNA synthesis on DNA supercoiling, III. Characterization of the inhibition of replicative and repair- type DNA synthesis by novobiocin and nalidixic acid, Biochim. Biophys. Acta, 653, 248-258.

Miller, K.G., L.F. Liu and P.T. Englund (1981) A homoge- neous type II DNA topoisomerase from HeLa cell nuclei, J. Biol. Chem., 256, 9334-9339.

Miller, M.R., and D.N. Chinault (1982a) Evidence that DNA polymerases a and fl participate differentially in DNA repair synthesis induced by different agents, J. Biol. Chem., 257, 46-49.

Miller, M.R., and D.N. Chinault (1982b) The roles of DNA polymerases a, fl, and "/ in DNA repair synthesis induced

251

in hamster and human cells by different DNA damaging agents, J. Biol. Chem., 257, 10204-10209.

Miller, M.R., J.J. Castellot Jr. and A.B. Pardec (1978) A permeable animal cell preparation for studying macro- molecular synthesis. DNA synthesis and the role of de- oxyribonucleotides in S phase initiation, Biochemistry, 17, 1073-1080.

Mosbaugh, D.W., and S. Lima (1983) Excision repair and DNA synthesis with a combination of HeLa DNA polymerase fl and DNase V, J. Biol. Chem., 258, 108-118.

Moses, R.E., and C.C. Richardson (1970) Replication and repair of DNA in cells of Escherichia coil treated with toluene, Proc. Natl. Acad. Sci. (U.S.A.), 67, 674-681.

Nishida, C., S.-Y. Choi, J. Kim, S. Keeney and S. Linn (1988a) DNA polymerase ~ plus HeLa- or human fibroblast cell- free extracts complement permeabilized xeroderma pigmentosum (XP) fibroblasts: Application for purification of XP correcting factors, in: E.C. Friedberg and P.C. Hanawalt (Eds.), Mechanisms and Consequences of DNA Damage Processing, Liss, New York, pp. 337-341.

Nishida, C., P. Reinhard and S. Linn (1988b) DNA repair synthesis in human fibroblasts requires DNA polymerase 8, J. Biol. Chem., 263, 501-510.

O'Connor, T.R., and J. Laval (1989) Physical association of the 2,6-diamino-4-hydroxy-5N- formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites, Proc. Natl. Acad. Sci. (U.S.A.), 86, 5222-5226.

Prelich, G., and B. Stiilman (1988) Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA, Cell, 53, 117-126.

Prelich, G., C.-K. Tan, M. Kostura, M.B. Mathews, A.G. So, K.M. Downey and B. Stillman (1987a) Functional identity of proliferating cell nuclear antigen and a DNA polymerase-8 auxiliary protein, Nature (London), 326, 517-520.

Prelich, G., M. Kostura, D.R. Marshak, M.B. Mathews and B. Stillman (1987b) The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro, Nature (London), 326, 471-475.

Pringle, J.R., and L.H. Hartwell (1981) The Saccharomyces cerevisiae cell cycle, in: Strathern et al. (Eds.), Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheri- tance, Cold Spring Harbor Laboratory, pp. 97-142.

Reinhard, P., P. Maillart, M. Schluchter, J.R. Gautschi and R. Schindler (1979) An assay system for factors involved in mammalian DNA replication, Biochim. Biophys. Acta, 564, 141-153.

Roberts, J.D., and M.W. Lieberman (1979) Deoxyribonucleic acid repair synthesis in permeable human fibroblasts ex- posed to ultraviolet radiation and N-acetoxy-2- (acetylamino)fluorene, Biochemistry, 18, 4499-4505.

Seki, S., and T. Oda (1988) An exonuclease possibly involved in the initiation of repair of bleomycin-damaged DNA in mouse ascites sarcoma cells, Carcinogenesis, 9, 2239-2244.

Seki, S., S. Mori and T. Oda (1989) ADP effects on bleomycin-induced DNA repair synthesis and adenylate kinase activity in permeable mouse sarcoma cells, Biochem. Int., 18, 235-242.

252

Sitney, K.C., M.E. Budd and J.L. Campbell (1989) DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene, Cell, 56, 599-605.

Smerdon, M.J., and M.W. Lieberman (1978) Nucleosome re- arrangement in human chromatin during UV-induced DNA repair synthesis, Proc. Natl. Acad. Sci. (U.S.A.), 75, 4238- 4241.

Smith, C.A., and P.C. Hanawalt (1978) Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 75, 2598-2602.

Smith, C.A., and D.S. Okumoto (1984) Nature of DNA repair synthesis resistant to inhibitors of polymerase a in human cells, Biochemistry, 23, 1383-1391.

Snyder, R.D. (1984) The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea, Mutation Res., 131, 163-172.

Syvaoja, J., and S. Linn (1989) Characterization of a large form of DNA polymerase 8 from HeLa cells that is insensitive to proliferating cell nuclear antigen, J. Biol. Chem., 264, 2489-2497.

Syvaoja, J., S. Suomensaari, C. Nishida, J.S. Goldsmith and S. Linn (1990) DNA polymerases a, 3, and c: Three distinct

enzymes from HeLa cells, Proc. Natl. Acad. Sci (U.S.A.), in press.

Tan, C.-K., C. Castillo, A.G. So and K.M. Downey (1986) An auxiliary protein for DNA polymerase-8 from fetal calf thymus, J. Biol. Chem., 261, 12310-12316.

Tanaka, K., M. Sekiguchi and Y. Okada (1975) Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendal virus), Proc. Natl. Acad. Sci. (U.S.A.), 72, 4071-4075.

Tanaka, K., H. Hayakawa, M. Sekiguchi and Y. Okada (1977) Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo, Proc. Natl. Acad. Sci. (U.S.A.), 74, 2958-2962.

Topal, M.D. (1985) Mutagenesis by incorporation of alkylated nucleotides, Basic Life Sci., 31,339-351.

Wahl, A.F., J.J. Crute, R.D. Sabatino, J.B. Bodner, R.L. Mar- raccino, L.W. Harwell, E.M. Lord and R.A. Bambara (1986) Properties of two forms of DNA polymerase ~ from calf thymus, Biochemistry, 25, 7821-7827.

Wong, S.W., J. Syvaoja, C.-K. Tan, K.M. Downey, A.G. So, S. Linn and T.S.-F. Wang (1989) DNA polymerases a and are immunologically and structurally distinct, J. Biol. Chem., 264, 5924-5928.

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A Critical Review of PermeabilA critical review of permeabilized cell systems for studying mammalianized Cell Systems for Studying Mammalian