The XPD complementation group

Mutation Research, DNA Repair, 273 (1992) 97-118 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8777/92/$05.00

97

MUTDNA 00182

The XPD complementation group Insights into xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy R.T. Johnson and Shoshana Squires Cancer Rt'scatch Campaign, Mammalian Cell DNA Repair Research Group, Departmou of Zoology, Unicersityof Cambridge, Cambridge CB2 3EJ (Great BritabO (Accepted 20 May 1991)

Keywords: XPD complementation group; Xeroderma pigmentosum, group D; Cellular DNA repair, defects in; Neurological abnormalities: Trichothiodystrophy; Cockayne's syndrome

Summary The xeroderma pigmentosum complementation group D is defined by more than 30 unrelated individuals of whom less than half show major abnormalities of the central nervous system, once considered to be the hallmalk of the group. Fibroblasts from the great majority of these individuals show very considerable sensitivity to UV light in vitro despite the fact that the cells carry out what appears to be substantial excision repair, as judged from repair synthesis and incision activity. This article reviews the XPD group and the defects in cellular DNA repair and examines the lack of correlation between repair and the appearance of neurological abnormalities. The article also discusses the recent awareness that at least some members of two other inherited conditions, trichothiodystrophy and Cockayne's Syndrome, carry mutations in the XPD gent.

By the mid 1970s the XPD complementation group was established on the basis of a few individuals all of whom displayed severe photosensitivity, tumours and marked neurological prcblems (Robbins et al., 1974; Cleaver and Bootsma, 1975). Since then there has been a steady increase in new XPD cases, but many of those affected individuals do not conform to the original pattern. As a group we now see that XPD individuals show great variety in the severity of their condition in terms both of tumour incidence and in abnormalities of the central nervous system. Moreover,

Correspondence: Dr. R.T. Johnson, Cancer Research Campaign, Mammalian Cell DNA Repair Research Group, Department of Zoology, University of Cambridge, Cambridge

CB2 3EJ (Great Britain).

defects or deficiencies in the immune response are becoming evident in this group. At the cellular level XPD has attracted much attention bccause great UV sensitivity is coupled to moderate and in some cases considerable repair synthesis. in addition to presenting new data on repair in some of the XPD mutants the purpose of this survey is to examine the diversity of cellular defects associated with this complementation group. Sadly, however, analysis of XPD mutants is as yet spnradic; very few have been the subject of wide investigation and the picture is therefore fragmentary. The recent demonstrations that fusions between XPD cells and cells from certain individuals with trichothiodystrophy (TTD), or XPH fail tO result in complementation of nucleotide excisiGn-repair defect suggests that mutations in the

98

XPD gene are responsible for several inherited human diseases (Wood, 1991). Each of these conditions, Cockayne's syndrome (expressed by XPH individuals), trichothiodystrophy and xeroderma pigmentosum (XP) are photosensitive, but only in XP (and in some CS in XP individuals) is this associated with the early onset and abundance of tumours in sun-exposed skin.

Abundance and distribution of XPD individuals Table 1 provides information about the abundance and geographic distribution of the three most common excision-defective XP complementation groups, A, C and D. These data, though almost certainly incomplete, comprise a reasonable guide to the occurrence of these mutations, For the XPD group the data represent unrelated individuals, and it is clear that their distribution is uneven with Europe providing more than two thirds of the known cases. Even excluding the German collection, Europe still provides more than half the individuals. This distribution contrasts sharply with that from Japan which is rich

TABLE I GEOGRAPHIC DISTRIBUTION OF THE MOST COMMON EXCISION DEFECTIVE XP COMPLEMENTA.

TION GROUPS"

North-America Europe excluding Germany

Germany lapan [~gypt

xp group A C 3 5 10 14 2 7 21 i 7 I2 43 39

Db 6~ 14 ~ 12 3~ 35

Adapted from Fischer et al. (1982), h D mutations from unrelated families; does not include the 8 independent TTD type 2 or 2 TTD type 3 mutations, " Tolal number of cases 7; 1 sib pair. d Tolal number of cases 17:3 sib pairs, including the previously designated XPH sibs who also show Coekayne's Syndrome features and the recently assigned XPD-CS individual XPgBR. c Total number of cases 4; 1 sib pair,

in XPA patients but where only three unrelated XPD families have been identified (Ichihashi et al., 1988). Nomenclature of XPD strains is provided in Appendix Table 1. The limited association between XPD mutations and severe neurological abnormalities In the early days of XP work it seemed clear that individuals falling into the XPD group were uniformly afflicted with severe neurological abnormalities (Robbins et al., 1974; Cleaver and Bootsma, 1975), sharing this characteristic with XPA individuals. Fibroblasts from XPA and D also proved to be more UV-sensitive than from other XP groups (Andrews etal., 1978). However, as Table 2 shows, the tight XPD association with abnormal neurology is now much less convincing (Pawsey et al., 1979). Taking the 35 XPD cases where data have been given more than half of the individuals show normal rather than abnormal neurology. Excluding the more recently charaeterised Mannheim group the proportion of neurologically normal individuals is 40%. The two children sharing features both of Cockayne's syndrome and XP, previously classified as belonging to a separate XP complemcntation group, XPH, display neurological symptoms associated with CS rather than with XP (Lafforet and Dupuy, 1978). It is worth pointing out that on diagnosis of XP a number of the individuals without apparent neurological symptoms were very young (Table 2) and they may therefore develop late onset CNS abnormalities (Kraemer and Slor, 1985). The XPD mutation is clearly associated with sun-induced skin tumours in many of the individuals though in a few, possibly because of effective sun screening, no tumours have been recorded (Table 2). It is also becoming clear that the quality of immune response may be an important consideration in the development of skin tumours (Bridges, 1981; Lehmann and Norris, 1989). Very few XPD individuals have been examined for the normality of their immune system (Table 2) but all that have show some unusual feature (Lafforet and Dupuy, 1978; Norris etal., 1990). Only two of these four individuals, however, are known to have developed skin tumours.

99

Verification of XPD mutations Traditionally XP cells have been placed into different complementation groups on the basis of short term studies of the recovery of normal levels of unscheduled DNA synthesis after U v challenge (Robbins et al., 1974). However, the work of Giannelli et al. (1982) indicates that caution must be exercised in the analysis of XPD complementation because this is slc,,v and may not reach normal levels of UDS. Alternative short-term assays for establishing complementation groups include recovery of growth potential of population containing fused cells after UV (Cleaver, 1982; attribution of XP1PO and XP108LO as XPD via lack of ability to complement XP2NE), or recovery of unscheduled DNA synthesis in XP x XP fused populations (Lchmann and Stevens, 1980; attribution of XPIDU via lack of ability to complement XP1BR). In many cases it is difficult to identify which XPD strains have been used to verify the status of others in UDS or other assays. Table 3, which is almost certainly incomplete, lists XPD and TTD provenance (in those TTD cases which carry the XPD mutation). A full list of XPD attribution is desirable,

Given some of the difficulties of measuring complementation by UDS in XPD (slew kinetics, sometimes very high levels of repair synthesis) an alternative route is by means of generating permanent hybrids between different XPD mutants. This can only be achieved, however, if one of the partners is a permanent cell line. There are 3 XPD lines, the SV4O transformed XP6BE and XP3NE (NIGMS Catalogue of Cell Lines 1990/1991) and the UV sensitive XP102LO - HeLa hybrid (HD2) (Johnson et al., 1985). Indeed, it was because of the unusual origin of HD2 that c r o s s e s w e r e made with other XPD fibroblasts (and w i t h X P I 0 2 L O itself) t o e s t a b l i s h its genetic identity (Johnson et al., 1985). Hybridisation with fibroblasts from each complementation group subsequently established that XPH could not be complemented by XPD and therefore should be included in the XPD group (Johnson et al., 1985, 1989). Permanent hybrids offer several advantages in the study of complementation. First they allow

the biological significance of repair to be established, and second, a wider range of repair chatacteristics can be analysed than is possible in short-term heterokaryons (Johnson et al., 1989; Johnson, 1989 and Fig. 1). Judged by complementation for UDS HD2 × XPH or x XPD heterokaryons could be considered positive, although the extent of improvement was slight compared with crosses involving fibroblasts from other complementation groups (Johnson et al., 1985, 1989). Perhaps the XPD x XPD improvement in UDS reflects a degree of intragenic complementation, though as might be expected the resulting proliferating hybrids did not show iraproved UVresistance.

20

~s

~ ~-g ~g ~~ o ,. ~

/~-

~

*..... jr.-" .. ,, ...;..-~'~':-"". . . . . . . . . =~""":-~-2-.. /~.7~..¢:':,, 4~~ ,~i/

10

I i / _ ~ ~ ' "

'. : e / ~ 0J ~ 0 2

' 4

' e

' o

uv dos, I Jm "~j Fig. 1. Accumulation of DNA breaks over 40 rain after irradiation as a funclionof UV dose in wild-typefibroblasts. [3H]Thymidine-prelabelled culturesfrom (48BR(o o): BCL-DI ( u - - t , I ; XPD heterozygotc fibroblasts. XPHI02LO ( • .... • ) : CRL 1159 (,~ .... • ) : CRL 1202 (0 .... , ) . and in hybrids produced between the permanent XPD cell line HD2TG (ra ra) and either XPD fibroblasts XPIIlLO (3/ll/e, • • ) : XPI07LO ( 3 / X F . , ,). or trichothiodystrophy fibrGblasts T T D 2 G L (3/GL2CCI. • Ill) were plated at 2 x 10"~ cells per 35-ram dish one day before irradialion. The cells were incubated for 40 rain before and after UV-irradiation with hydroxyurea and ~,:osine arabinoside. After the 30-rain postirradiation period the cells were lysed in alkali and the DNA eluted from hydroxyapatite columns to determine the frequency of repair-related DNA breaks. For other details see Squires and

Johnson(1988).

100 T a b l e 4 shows t h e c u r r e n t s t a t u s o f f u s i o n s involving XPD x XPD partners. These studies w e r e u n d e r t a k e n to verify the X P D s t a t u s o f several X P D fibroblast strains a n d one T T D strain

in p r o l i f e r a t i n g hybrids. F o r t u n a t e l y t o d a t e all crosses i n v o l v i n g D × D f u s i o n s h a v e f a i l e d to g e n e r a t e h y b r i d s t h a t have r e c o v e r e d U V resist a n c e o r e x c i s i o n r e p a i r ability.

TABLE 2 CLINICAL SYMPTOMS IN XPD INDIVIDUALS Individual I

Sex

Age

Neurological problems 2

XPIPO " ~"XP5BE b S L XP6BE h XP7BE b XPI02LO ~ XPI04LO ~ I XPI07LO c

M F F F F F M

2 26 20 !I 18 !I 5

Yes. severe Yes. severe Yes. severe Yes. severe No No 3 No

F

aborted

S

Immune systems

Turnouts 4

T b T h T b none c none c < 50~ of normal NK cytotoxicity d

L XPI08LO ~

XPII I LO c

M

7

No

< 50% of normal NK

T c.d

cytotoxicity d

XPI35LO d

M

7

XPIBR ~ XPIKC" XPKABE ,.t

F F F M F M F

6 9 22 23 28 28

M F

I0

X P I N E "'"

I XP2NE "'~ XP3NE ~`" XPI DUh XPlSPV t XPI6PV * XPl7PV S I XP-CS-2 J "Patient 7" J XP8BR k XPI0TO I XP43KO m r XP58TO m. S L XP59TO m. XPITBE h S

XP9MA a XP 12MA a XP 17MA ~ XPI8MA a xPIgMA ~ XP33MA " XP36MA ~ XP39MA p XP40MA p XP46MA p XP47MA p XP55MA q

M F M F M F M F F

M

< 50% of norm~.l NK cytotoxieity d

none d

Yes. severe

2 2 51 31 8 6

Yes. severe Yes, severe Yes. severe Yes. severe

Yes. CS-like Yes. CS-like Yes. CS-like No

No Borderline No

14

Yes. severe

11 42 I1 38 33

No No No No No No

T T

Diminished PHA response

T none T T none none T T

none none T

I01 Repair defects in XPD

(I) UV-sensitit'ity offibroblasts and limited host-cell reactivation of irradiated viruses The early work of Robbins et al. (1974), Kraemer et al. (1976) and Andrews et al. (1978) estabfished that XPD fibroblasts were extremely and uniformly UV-sensitive, second only to XPA cells and substantially more sensitive than XPC. Excepting some of the cell strains from the Mannheim collection which arc unusually resistant to ultraviolet (Appendix Table 3) (and express considerable excision repair ability), all reports (Appendix Table 2) indicate substantial killing by U V with virtually no shoulder on the survival curve. Compared with XPA cells, however, when quiescent holding is imposed after UV, X P D fibroblasts (XP7BE, XP3NE, XPKABE) show improved survival in line with their very limited repair capabilities (Simons, 1979; Kantor and Hull, 1984). Reactivation of U V damaged adenovirus 2, in X P D cells, assayed by the production of viral antigen (Rainbow, 1980) or viable virus (Day, 1976; Abrahams and van der Eb, 1976; Arase et al., 1979) has been examined in very few mutants [XPI02LO, through its derived hybrid HD2, Johnson et al., 1985; XPSBE, XP6BE (sibs) and

XP7BE (Day, 1976; Abrahams and van der Eb, 1976; Arase et al., 1979)]. In each of these strains repair of damaged virus is greatly depressed compared to wild-type cells (Appendix Table 2). Enhanced reactivation of damaged virus, an indicator of an inducible excision-repair response, has been observed in one XPD strain (XP3NE) for Herpes simplex virus type I (Abrahams et al., 1984). These studies report a similar time scale of enhanced reactivation in XPD, XPC and XPA cells as is found in normal cells, and also a similar magnitude. For XP cells, however, the amounts of UV given to the cells and to the virus were considerably less than for WT (8-fold and 4-fold respectively).

(2) UV-induced incision activity in XPD cells XP cells are deficient in one or more early steps of the excision-repair pathway. The ratelimiting UV-induced incision step is determined most readily by the use of repair-synthesis inhibitors, where incomplete repair sites accumulate as D N A breaks. These breaks are quantitared by means of alkaline unwinding followed by centrifugation in alkaline sucrose gradients or by hydroxyapatite chromatography (Collins and Johnson, 1984). The time course of UV-induced excision repair consists of two kinetics; a rapid

Notes to table 2 t S indicates affected sibs,

a blank indicates not known, •~ slightly deaf. 4 blank indicates not known, '~ NIGMS Databank. Robbins et al. (1974); Andrews et al. (1978). c Pawsey et al. (19"/9). d Norris el al. (1990), patient I, XPI35LO; patient 4, XPI07LO; patient 5, XPIIILO. e C.F. Arlett, personal commuaication. t ATCC Database. Thrush et al. (1974). h B. Johnson, personal communication. i M. Stefanini, personal communication. J Lafforet and Dupuy (1978). k D. Bootsma and A. Lehmann, personal communication. t Takebe et al. (1977); Mamada et al. (1988). m Ichihashi et al. (1988). " Fujiwara and Sato (1985). o Fischer et al. (1982). P Thielmann et aL (1985). q Thielmann et al. (1986).

102 one during the first 6 h after irradiation and a slower one that continues for at least a day. Table 5 and Fig. 2 provide comparative data

higher initial rate of arrested repair-site accumulation, and at low U V doses accumulate D N A breaks at frequencies similar to normal cells. On

for the early incision activity in a selection of XPD strains immediately after UV-irradiation. Fig. 2 shows that in the presence of DNA synthesis inhibitors the accumulation of DNA breaks during the first 40 min after UV is dose-dependent. The dose-response curves of break frequencies in the majority of the XPD strains are very similar, the number of inhibitor-arrested repair sites increasing to a maximum of around 3 breaks/10 ') dalton, which is only 10-20% of the normal level (Table 5; Appendix Table 2). There are a few exceptions which show significantly higher frequencies of DNA break accumulation. XP-CS-2 cells (previously assigned XPH) show a

the basis of its incision kinetics the XP-CS-2 cell strain was considered to be quite distinct from the 'typical' XPD cells (Squires and Johnson, 1988). The other mutant, XP1PO, can be singled out as the most unusual XPD cell strain. It shows a high level of UV-induced incision, about 60% of that of normal, as measured by DNA break accumulation and UDS (Fig. 2 and Table 5). It has been reported that XP1PO has about 2 / 3 of wild-type ability to remove angelicin monoadducts (Cleaver and Gruenert, 1984) and about 40% of normal UV induced UDS (Cleaver et al., 1984). A detailed kinetic and biochemical analysis of repair in XP1PO cells is now in progress.

TABLE 3 ATTRIBUTION OF XPD OR TTD MUTANTS BY HETEROKARYON ASSAY OF UNSCHEDULED SYNTHESIS OR REPLICATION RECOVERY AFTER UV Designated XPD mutation XPSBE (XP6BEsib)

Fusions for attribution The initial XPD strains;

XPTBE

will complement XPA, B and C but not each other

XP$BE

XPIO7LO

Author Robbins el al. (1974)

Pawsey cl al, (1979)

XPlllLO XP3NE. XP2NE XP3NE

Identifiedas X P D unpubllsh~ddata of E. de W¢~rd-Kastel¢in,W, Keijzer and D, Bootsma citedin Dc W¢¢rd-Kasteleinet al.(1976)

XP3NE

XPI02LO XPI04LO XPI08LO

XPIBR

XPIDU

Lehmann and Stevens(1980)

XP2NE

XPIPO XPI08LO

Cleaver (1982)

XPS9TO

XP43KO

lchihashi et al. (1988)

XPKABE and XPITBE

Identified as XPD, data unpublished(Andrewset al., 1978)

XP3NE

"VrDI PV TTD2PV

TTD 1PV

TTD2PV TTD4PV TTD4PV

"I~D4PV

TTD2GL

Lehmann et al. (1988)

TTD4PV

TTD1BI

Lehmann et al. (1988)

Stefanini el al. (1986)

103 TABLE 4

8

.

COMPLEMENTATION ANALYSIS USING PROLIFERATING HYBRID CELLS -

Permanent XPD line HD2TG and

Hybrids with no recovery of UV R

Hybrids with recovery of UV R

XP102LO sJ XPIBR sJ XP1NE s XPI07LO XPIIILO XP6BESV s XP-CS-2 sj "VI'D2GL s,t XPI02LO s

and XPA, B, C, E, F and G

6

/

~

SJ

XP6BESV and

I

-

~~-~

m o~

and XPI s (i.e. XPC), XPA (XPSV2OS) s

i

. , r 4

a o Q. v

HD2TG s

~

XP-CS-2 XPIPO s s XP7BE s

Fig. 2 shows the time courses of DNA break accumulation of UV-irradiatcd fibroblasts of normal, XPI PO, XP-CS-2, and two representitives of the low incision activity XPD group, XPIBR and XP135LO. In the normal and XPIPO cells, in the continuous presence of DNA-synthesis inhibitors, D N A breaks start to accumulate immediately af-

,

0

-

-

~

~

, ~

0~ 0

t

2

4

6

Fig. 2. Accumulation of DNA breaks as a function of UV dose in XPD fibroblasts. XPIPO (D C3): XP-CS-2 to--o): XPI07LO (& z~), XP7BE

(,

,): XPIDU (11 XPIBR: ( • ~

•

ii): XPI35LO (<~ )"TTD2GL(X).

XPIPO XP-CS-2 XP5 or 6BE XP 102LO XP3NE

0.47 0.4 0.6 0.39

% of qormal cyclobutane dimer repair, 24-48 h

5-10 0 15

For fuller details and references see Appendix Table 2.

,'~l:

ter UV-irradiation but at a non-linear rate i.¢. the rate diminishes with time. in the other XPD cell strains, XP-CS-2, XP1BR and XP135LO, the ini-

CHARACTERISTICS OF UV-INDUCED REPAIR IN SELECTED XPD CELL STRAINS Do (UV Jm- 2 )

8

UV d o s e ( J m "2 )

TABLE 5

Skin fibroblasts from

,

f ~

2

s Colony survival. I Incision activity. All fusions listed excepting HD2TG x XPI07LO, XPOI I ILO and TI'D2GL and XP6BESVxXPIPO and XP7BE were reported in Johnson etal, (1985, 1989}. In the HD2TG fusions hybrids were selected in HAT, clones picked and tested for UV-resistance and incision ability (Fig. 1). In the XP6BESV fusions clones were tested for UV resistance from those t h a t grew up alter a UV dose of 3 Jm -2.

,

~ of normal (6-4) adduct repair at 6 h

< 10 15

~,~ normal incision 2 Jm - 2, 40 rain

Repair synthesis e/~ normal

58 3i 23 21

65-66 40-52 21-23 20 18-28

11)4

2o

XPD cells from the Mannheim Collection

f

1~ ...............-o

•a=i m= es to ,n= ze s =,-. o.

•..

ss

01 0

~......za..........~_ ~ ~-'-'~,~

20

40

60

I '

s0

Incubation time ( r a i n )

The Mannheim Collection of cell lines, clinical data and repair characteristics from XP individuals represents an important addition to the field. The bulk of the data are published in four papers (Fischer et al., 1982; Thielmann et al., 1982, 1985, 1986) and describe approximately 50 new XP cell lines, among which are 12 XPD examples (Table 2 and Appendix Table 3). Clinical descriptions are limited to 5 individuals none of whom show abnormal neurology; two of these including a woman of 38, had no history of skin tumours. As might be expected the 42-year-old sailor, XP12MA, has had many skin tumours. The most unusual feature of this XPD collection is the presence of several very UV-resistant strains (XP12MA, XP17MA, XP46MA), one of which (XP12MA) has near normal repair ability (Appendix Table 3). In the absence of complementation data it is hard to see how XP12MA could have been designated XPD, and information about the control XPD strains used to verify the status of new strains is not given. A further

Fig, 3, Time courseof DNA break accumulation in UV-irradinted (4 Jm --~) normal (48BR, o ~ o , BCL-DI,

difficulty is the occasional disparity between repair data obtained by means of different assays

t, ~ ) fibroblasts and XPD fibroblasts ( • ~ •, X P I B R ; [] ~ e~. XPIPO; , ' o ~ o . XPI35LO; • - - • . XP-CS-2). Experimental details are given in Ihe

on

legendtt, Fig I.

the same cell strain e.g. XP33MA, which shows low UDS but extremely high incision ability (Appendix Table 3), Despite these problems the Mannheim XPDs represent a valuable resource for future work on this complementation group, Removal of UV-induced DNA lesions In XPD cells

tial rate of break accumulation is much slower, but it is almost linear for long periods, and, given enough time, the number of DNA breaks aecumulated reaches about 40% of that of the normal cell. These and earlier results (Squires and Johnson, 1988) indicate that in the majority of the XPD cell strains the level of repair enzyme(s) is about 10% of normal, in a study of some of the XPD cells (XP102LO, XPI04LO, X P I l l L O ) Pawsey et al. (1979) showed that the time course of UDS was characterized by a persistent steady increase up to 6 h, reminiscent of the time-dependent increase in incision activity we find for these cells. A similar steady increase in UDS has also been reported for XP43KO by Hiramoto et al. (1989).

Incision activity and repair synthesis are legitimate indicators of excision repair but need to be judged in relation to the actual loss of photochemical lesions. Can the rather high levels of apparent repair in some of the XPD cells be squared with lesion removal, and if so how does this accord with the great UV sensitivity of the cells? As yet there are few answers to these questions, and few XPD strains have been studled. There are no cyclobutane (CB) dimer or (6-4) adduct removal data for any of the most "repair active" XPD strains (XPIPO, XP-CS-2) where at least some (6-4) adduct removal might be expected from the early wave of incision and the high UDS levels. A comprehensive compari-

105 son of the UV-excision-repair capabilities, as measured by cell survival, DNA break accumulation, UDS, CB and (6-4) ad,~luct removal, and apurinic endonuclease activity is shown in Appendix Table 2. CB direct repair analysis has been restricted to 4 different cell strains, or 5 if type II trichothiodystrophy is included (Table 5, Appendix Table 2). Assays based on dimer endonuclease sensitive sites remaining in the DNA, or anti CB dimer antibodies, in combination with low UV fluences, have generated the most useful data. Using these techniques CB dimer loss over 24 h ranges from 0 to 10% of normal after 10 Jm -2 (XP102LO, XP6BE) (this paper; Mitchell and Nairn, 1989) 10% of normal after 40 Jm -2 (XP43KO, Hiramoto et al., 1989), less than 20% of normal (TTD2GL) after 8 Jm -2 (Broughton et al., 1 9 9 0 ) and 20% after 2.9 J m - 2 (32 h) (Zeile and Lehman, 1979). The most detailed study of dimer loss in XP cells by Kantor and Hull (1984) examined lesion removal by the dimer endonuelease procedure in quiescent fibroblasts over a period of 50-70 days after i Jm -2. Both XPD mutants tested (XPSBE and XPKABE) showed similar biphasic repair profiles to normal fibre)blasts although both fast and slow rates for XPD were around 10-fold less. Given sufficient time XPD cells seem able to eliminate all cyclobutane directs, unlike either XPA or XPC cells. Typical XPA leave a residual level of 0.3 direct sites per 10H dalton (about 1000 per genomc); XPC, how. ever, do not express the slow repair component and leave a residual 25-30% of directs in the genome (Kantor and Hull, 1984). The slow, steady and complete repair of cyclobutane directs found in XPD under non cytotoxic conditions indicates that the product of the D locus, already shown by Giannelli et al. (1982) to be present in limiting amounts in the normal partner of WT × XPD heterokaryons, is quantitatively reduced by at least an order of magnitude in some XPD mutants, The XP102LO-derived hybrid cell, HD2, which expresses the XPD defect is somewhat less UVsensitive than its parent fibroblast (Johnson et al., 1985), though it is still unable to complement different strains of XPD fibroblas~s (Johnson et al., 1985, 1989, this paper). Examination of its CB

dimer removal capability by means of monoclonal antibody assay after 10 Jm -2 shows that it removes more lesions than XP102LO, though it is still much worse than a normal cell (Table 5). In the past few years the pyrimidine (6-4) pyrimidone photoproduct has emerged as a major ultraviolet lesion in DNA, responsible with CB dimers for cell killing, arrest of replication, and for mutagenesis (Mitchell and Nairn, 1989). In all normal cells yet examined, (6-4) photoproducts are removed with much greater speed and cornpleteness than the cyclobutane dimers. By 6 h 90-100% of these adducts have been removed and, at least in Chinese hamster cells, they appear to be even more rapidly lost from transcribing genes than from bulk DNA (Thomas et al., 1989). For the first 6 h after UV-irradiation the intense excision-repair activity is likely focusse0 on (6-4) photoproducts rather than on CB dimers as judged from studies with mutant hamster cell,, (Thompson et al., 1989; Zdzienicka et al., 1988) and type 3 trichothiodystrophy fibroblasts (Broughton et al., 1990), which have, respectively normal (6-4) photoproduct removal but no CB repair and normal CB dimer repair but limited (6-4) removal. Following the initial wave of (6-4) repair (Mitchell and Nairn, 1989)incision activity rapidly declines, presumably reflecting the less active repair of CB dimers. Only three XPD mutants have been surveyed for (6-4) adduct loss and all are grossly defective. 6 h after 10 J m - : UV XP6BE removes about 25% of the (6-4) lesions (Clarkson et al., 1983); XP102LO about 15% (Mitchell and Nairn, 1989; Table 5); by 24 h about 50% of (6-4) adducts are removed from XPI02LO (Table 6). Over 24 h XP43K0 removes 10% of the (6-4) adducts after 40 J m - : (Hiramoto et al., 1989). The UV-sensitive hybrid HD2, derived from XPI02LO also shows poor removal of both cyclobutane dimers and (6-4) adducts. Compared with parental XPI02LO, however, repair of both lesions is somewhat improved (cyclobutane dimers: 57% remaining at 6 h compared with 100%; (6-4) adducts: 52% remaining at 6 h compared with 86%, Table 6). This improved repair is reflected by the improved survival after UV (Johnson et al., 1985). There has been much interest in the reiatknship between trichothiodystrophy (TTD) and XP

106 TABLE 6 (A) TIME C O U R S E O F T H E R E M O V A L O F C Y C L O B U T A N E DIMERS IN D N A A F T E R i0 Jm -2, ASSESSED BY A N T I D I M E R ANTIBODY % of initial cyclobutane dimers remaining after UV (h) 1.5

Cell normal human fihroblast " HeLa h

XPI02LO h HD2 h XP6BE a

75 85+ 13 117+_ 6 q 3 + 10 > 90

3

6 50 89_+5

> 90

50 6 9 + 10 102+- 15 57+- 4 > 90

24 40 94_+7 41+-5 > 90

(B) T I M E C O U R S E OF T H E R E M O V A L OF (6-4) A D D U C T S IN DNA A F T E R 10 J m - 2 ASSESSED BY ANT! (6-4) A D D U C T ANTIBODY % of initial (6-4) adducts remaining after UV (h) 1.5

Cell normal human fibroblast " HeLa h

XPI02LO t, HD2 h XP6BE "

25 37+-8 103.4-8 102+-2

3

6

10 84- 1 87+- l l 77+- 4 > 90

5 3 86+-5 6~-t-6 > 90

24 3 47+-4 80

" Mitchell el el, (1985); Mitchell and Nairn (1989), h Dtua o f D,L. Mitchell and R, Nairn, representing the mean and SE of three determinations,

with the demonstration that fibroblasts from eertain TTD individuals are unable to complement repair synthesis in XPD fibroblasts (XP6BE) (Stefanini et el., 1986). Of the three types of TTD, identified on the basis of their cellular UV sensitivity and repair abilities, type 2 resembles XP6BE ~nd XPI02LO in its limited removal of CB dimers and (6-4) photoproduets over 6 h, while type 3 can remove CB dimers normally but is unable to remove more than 50% of (6-4) adducts over 6 h (Broughton et al., 1990). Since type 3 TTD ceils are unable to complement type 2 for repair synthesis (Lehmann et al., 1988) and since type 2 does not complement XPD, we see that the type 3 TTD repair phenotype (of which there are now 2 examples) represents the slightest repair modifying mutation at the XPD locus, Sensitivity of XPD heterozygote cells to UV and

DNA synthesis in several XPD heterozygotes compared with presumed homozygous normal individuals. We have examined UDS and initial incision activity in three heterozygote cell strains (XPHI02LO, the mother of XPI02LO, CRL 1159, mother of XP5 and 6BE and CRL 1202 the mother of XPTBE), and find these activities to be normal ( U D S ) o r slightly less than normal (tactsion) (Appendix Table 2 and Fig. 1). The differeaee in incision activity between normal and XPD heterozygote cells seen at higher UV doses perhaps indicates a lower level of enzyme. The lower incision activity of the D heterozygotes is not so marked as that found in A heterozygotes, which show exactly half the initial activity of normal cells (Squires and Johnson, 1988). Survival of the three D heterozygote fibroblast strains mentioned above to UV falls within the normal range (S. Squires, unpublished data).

their repair e~pability

Further comments on CB dimer removal in XPD

Little needs to be added to the survey carried out by Pawsey et al. (1979) who observed a slight though significantly reduced level of unscheduled

cells From the first XPD cells have been regarded as having unusually high levels of UV-induced

107

repair synthesis by comparison with other XP groups (Robbins et al., 1974). Some of the stocks held in the NIGMSH Human Genetic Mutant Repository are described as having near wild-type levels of unscheduled synthesis. However, with the exception of certain of the XPD strains in the Mannheim collection which are reported as having virtually normal repair synthes~s~ we find that only one other XPD mutant (XP1PO) shows greater than 50% of the wild-type level of UDS during the early period after UV (Table 5; Appendix Table 2). UV induces considerable repair synthesis in XPD cells, more than in XPC and much more than in XPA or XPG. Moreover the unscheduled synthesis is maintained for a considerable period. These considerations led Paterson and colleagues (Pate~'son, 1982; Paterson et al., 1984, 1987; Weinfeld et al., 1986) to propose that the XPD mutation results in the accumulation of an intermediate in the excision-repair pathway of CB dimers which in normal cells is rapidly processed and is therefore difficult to detect. The intermediate appears in 2 XPD sib pairs (XP2 and 3NE, XP5 and 6BE) as a non-photoreactivable dimer and over 48 h accounts for approximately 15% of the initial dimer yield. In XPA but not XPC cells the same intermediate appears, but is not associated with further repair action (i.e. incision, excision and repair synthesis). In XPD cells the novel modified dimers are suggested as the sites of abortive repair synthesis. Paterson's model implies that many of the modified dimer intermediates are not removed but merely serve as sites of abortive repair synthesis. However, as we have seen, XPD cells do show limited dimer removal which is biologically significant (Simons, 1979; Kantor and Hull, 1984), and further confirmation of the novel XPD intermediate and its role in normal repair is thus required, The careful quantitative work by Giannelli.and colleagues on complementation in heterokaryons (Pawsey et al., 1979; Giannelli et al., 1982) provided information about the nature of the gene product. Even in wild-type fibroblasts the D product is limiting and requires continuous protein synthesis to maintain adequate levels. Moreover, the product appears to accumulate in nuclei and only slowly equilibrates among the various nuclei of heterokaryons. In XPD mutants the

slow removal of dimers and the steady increase in repair activity after irradiation suggests that gaining access to dimers in chromatin may be a particular problem. Data provided by Smerdon et al. (1982) support this view. In this work sodium butyrate was used to hyperacetylate core histones as a potential means of increasing DNA repair. Acetylation has the effect of reducing the net positive charge of the histone and thus of pos~.~bly displacing the nucleosome from the DNA. The net effect of histone acetylation is to make the DNA more accessible (Turner, 1991). Similar levels of core histone acetylation in normal, XPC and XPD (XP6BE) fibroblasts resulted in increased repair synthesis over the first 4 h after UV, and this was especially marked in XPD (3-fold enhancement). Moreover, while histone acetylation was marked by enhanced repair synthesis in WT cells at early times, the net removal of dimers was not affected, unlike in XPD where sodium butyrate treatment resulted in a significant overall increase in dimer loss. The final consideration of dimer removal in XPD cells concerns the pattern of repair w are there indications of unusually restricted repair of dimer removal, such as those associated with nuclear matrix-bound transcribing genes in XPC (Mansbridge and Hanawalt, 1983; Mullcnders et al., 1984, 1986)? The answer seems to be no. Karentz and Cleaver (1986) examined the distribution of CB direct repair sites in XP1NE and XP1PO, comparing the pattern with normal and XPC cells. Their finding of randomized repair sites in XPD over a 24 to 48 h period strongly suggests that XPD expresses a general downgrading of repair throughout the genome, results in agreement with Mullenders' finding of a normal matrix-associated repair pattern, albeit a diminished one, in XPD cells (XP3NE) (L. Mullenders, personal communication). Apurinic endonucleases and XPD Not only is the nucleotide excision-repair mechanism defective in XPD but there is also strong evidence for an aberrant base-excision mechanism too, at least in some members of the XPD group. Removal of DNA damage, as after free radical attack, is carried out by DNA glycosy-

lases which remove the damaged base followed by apurinic (AP) endonuclease incision of the phosphodiester backbone, 3, or 5' to the damage site. Glycosylase and AP endonuclease functions are in some cases carried out by the same enzyme. In 1976 Kuhnlein et al. reported that several XP complementation groups (A, B, C, D and Variant) had altered AP endonuclease function. XPA and D were the most severe'~y affected groups and D cells had the least activity. Subsequent studies on XPA and C suggested that their AP endonuclease activities were normal (Helland et al., 1984), but further work on XPD had revealed that of the two species of AP cndonuclease separable by means of phosphocellulose chromatography from human cells, AP cndonuclease I was missing (Kuhnlein et al., 1978). AP endonuclease I proved to be a typical Class I AP endonuclease, mediating phosphodigster bond cleavage by means of a/3-elimination process and also having associated DNA glycosylase activities, AP endonuclease !I (a class I1 AP endonuclease), the more stable of the two enzymes, proved to be present at wild-type levels and with wild-type function in XPD ceils. However, it is now clear that absence of this enzyme is not an absolute diagnostic of the XPD group. Appendix Table 2 shows that API endonuclease is absent from XP7BE, the sibs XPSBE and XP6BE, and from XPIPO (S. Linn, personal communication) but is present in XPIBR (S. Linn, personal communication) and the XP102LO hybrid derivative HD2 (G.C. Elliott, R.T. Johnson, S. Linn, preliminary data). With the exception of XP102LO these XPD strains are derived from individuals with neurological abnormalities. There is, therefore, a less than perfect correlation between the lack of enzyme and CNS malfunction among XPD strains. All of these XPD strains, excepting XP1PO, show similar low levels of UV-induced incision events, and cells lacking AP! endonuclease activity fail to complement cells with the enzyme (XPI02LO(HD2)x CRL 1157; see below. Presence of the enzyme in extracts of certain XPD strains does not, however, prove that it is active in vivo and a functional assay for the enzyme is required. One attempt to relate the lack of API endonuclease in XPD to a biological

endpoint demonstrated instead that XPD repaired depurinated SV40 molecules much better than wild-type cells thereby achieving excellent host cell reactivation (Kudrna et al., 1979). Neither is there evidence of AP endonuclease I-XPD cells being unusually sensitive to alkylating agents, whose spectrum of damage usually includes considerable depurination (Thielmann et al., 1986). The participation of API endonuclease ~n the repair of cyclobutane pyrimidine dimers or (6-4) photoproducts in eukaryotic cells remains uncertain (see, for example Warner et al., 1980). Much more certain is the action of API endonuclease/ glycosylase enzymes on DNA heavily damaged by ultraviolet where they mediate removal of bases damaged by free radicals (Linn, 1982). Concluding remarks XPD mutations at the cellular level

At the cellular level all XPD mutants, including the most repair defective group of TTDs, are extremely UV-sensitive, and despite the limited amount of work on most of the mutants the majority appear remarkably similar in phenotype. XP7BE and XPI02LO represent this group and display limited removal of the major photoproducts. Repair continues slowly for many hours and is biologically effective. The general validity of abortive repair and non-photoreactivable CB dimers in XPD (and XPA) strains, described by Paterson and colleagues, remains to be proven as an important diagnostic of the mutants. As expetted, the limited removal of photoproducts renders XPD cells hypermutable by UV (XPSBE, Maher et al., 1979; XPTBE, GIover et al., 1979) and more readily induced by UV to undergo more neoplastic changes than normal fibroblasts (XP7BE, Maher et al., 1982). There are a few cellular oddities among XPD strains and these include XP-CS-2 (formerly XPH) which shows normal incision kinetics immediately after low UV doses, and XPIP0 which has surprisingly high and sustained incision ability, We might speculate that each of these mutants has more extensive (6-4) adduct repair than other D strains, but that such repair does not increase the degree of resistance to UV. Given the considerable heterogeneity of repair pheno-

109

type that is known to exist among certain hamster complementation groups (Collins and Johnson, 1987; Zdzienicka et al., 1988; Mitchell et al., 1989; Regan et al., 1990), the unusual XPD strains no longer appear extraordinary, though more detailed analysis of their UV-induced repair profiles would be interesting, Most unusual among the XPD mutations are those responsible for generating the repair-defecrive component of the third type of 1TD individuals, where normal CB dimer removal kinetics are observed, but which are defective on the early wave of (6-4) adduct removal. Even here, however, there are similarities to the overall slow XPD-repair phenotype, since given sufficient time normal amounts of (6-4) adducts are removed (Broughton et ai., 1990). This group of "ITD mutants also provides the strongest evidence that the XPD gene product may have separate domains for carrying out repair of different photoproducts, The role of aberrant base-excision repair, mediated by apurinic endonucleases/glycosylases, in the aetiology of XPD remains uncertain. The tight correlation between XPD and the absence of Class I AP. endonuclease (Kuhnlein et al., 1978) is no longer clear, though the in vivo functional status of the AP endonuclease I found in some XPD strains remains to be determined, Despite the difficulty in explaining how AP endonucleases with no CB dimer specificity participate in the removal of the major UV-photoproducts from eukaryotic DNA, it may be easier to imagine their involvement in the process than to postulate XPD as a double mutant, defective both in nucleotide and base excision repair pathways. That a Class I AP endonuclease/glycosylase, albeit one with CB dimer specificity, can stimulate repair and dimer removal in XPD cells has been verified by introducing either the phage T4 endonuclease enzyme directly into cells (Tanaka et al., 1975) or by transfer and expression of the T4 denV gene (Arrand et al., 1987). Finally, using a permeabilized cell system Linn and colleagues are providing evidence for the critical role of a Class I AP endonuclease/UV endonuclease II! in restoring normal DNA-repair synthesis to XPD fibroblasts, irradiated with a large UV dose (40 Jm -2) (Kim and Linn, 1989;

Keeney and Linn, 1990). Questions remain about the substrate specificity of the AP endonuclease in this system and whether it facilitates the operation of a much wider nucleotide excision-repair capability. Finally in the light of the XPH-XPD problem it is worth pointing out that complementation studies involving heterokaryons may give rise to unreliable data. Verification of membership of a particular complementation group can also be accomplished by the construction of permanent hybrid cells since almost all XP groups now inelude an immortal and verified cell line. Permanent hybrids allow a wide range of repair assays to be carried out to confirm complementation or the lack of it, and by means of DNA fingerprinting the provenance of the cells can be readily established. Cloning the XPD gene remains a major goal. The first steps have been taken and a Chinese hamster sequence isolated that confers a degree of UV-resistance on the XP102LO-derived hybrid HD2,and improved DNA repair (Arrand et al., 1989). XPD mutations at the level of the individual It is now apparent that a defect in the XPD gene can give rise to a much wider variety of clinical symptoms than was originally supposed. Setting aside the incidence of skin tumours for the moment less than half of the individuals diagnosed as XPD from UDS complementation assays display the severe degenerative disease of the CNS that was once considered the hallmar!¢ of the D group. To some extent the absence of neurological symptoms in some individuals m~y be related to their juvenile age since this is generally a progressive condition; nevertheless, we should not be too surprised to find wide variation in the clinical symptoms associated with (presumably) different mutations in the D gene. For example, mutations in different regions of the HGPRT gene are associated with quite distinct clinical symptoms - - mild to severe gout at one extreme, major neurological abnormalities of Lesch-Nyhan at the other (Melton, 1987). Similady, different mutations in the XPAC gene, responsible for the XPA condition, result in severe or mild forms of the disease (Tanaka et al., 1990).

110

The child designated XP1PO, whose repair characteristics are the most unusual of all the XPD mutations examined, showing the highest level of incision and repair synthesis activity (Table 5) marks the most extreme clinical symptoms. In addition to XP1PO's severe neurological abnorrealities, there are also serious developmental aberrations involving the gut and the family has a history of Charcot-Marie-Tooth Disease, an axonal type of peroneal muscular atrophy (NIGMS databank), Variable incidence of cancer among XPD individuals is presumably related in part to childhood sunscreening and subsequent lifestyle m the 42year-old sailor listed in the Mannheim Collection has many recorded skin tumours. There is increasing evidence, however, of impaired immune function among XP groups, including D, and the suspicion that for skin tumours to develop even in residually damaged XP tissues one or more elements of the immune response must be subnorreal (Burnet, 1971; Bridges, 1981; Norris et al. l989, 1990). The identification of an underlying XPD mutation responsible for the abnormal repair of some trichothiodystrophy patients (Stefanini et al., 1986) represents a remarkable and iiluminating extension of XPD clinical phenotypes. The absence of TTD symptoms (brittle hair, abhormalities of sulphur metabolism)among members of the 'classic' repair defective XPD group has prompted Lehmann and Norris (1989) to propose that the TTD and XPD genes lie close to one another; mutation in TTD alone generates repair competent TTD individuals but a mutation in TTD extending into the XPD gene produces TTD clinical symptoms allied to sun sensitivity and repair defect. Most important from the viewpoint of tumour aetioiogy are the observations that repair-defective TI'D individuals do not have an abnormally high incidence of skin cancer, and, in contrast to XPD patients, show normal immune response (Norris et al., 1990). Individuals with mutations in XPD may also express the clinical symptoms of Cockayne's syndrome, a conclusion arising from the inability of XPD cells to complement fibroblasts from the now defunct XPH group (Johnson et al., 1989, Vermeulen et al., 1991). The "XPH" (XP-CS-2~

mutation arose in a single family with two affected children (Table 2) who expressed syruptoms of both XP and CS. Initial complementation assays for UDS supported the creation of a new XP complementation group (Moshell et al., 1983; Robbins et al., 1983; Robbins, 1989; Johnson, 1989). Recently a second X P D / C S individual, XP8BR, has been identified (A. Lehmann, D. Bootsma, personal communication), and it is now clear that the hallmarks of CS at the level of the individual coincide with several XP complementation groups (XPB, Robbins et ai., 1974; Weerda et al., 1990), XPG (D. Bootsma, personal communication; Wood, 1991), as well as with XPD. CS, of course, also occurs in individuals without XP symptoms, where the effects of the mutation can be clearly discerned at the cellular level (Lehmann, 1982). In these individuals, however, there does not appear to be an exaggerated incidence of skin tumours (Lehmann and Norris, 1990). CS is clearly defined by mutations in several genes in addition to appearing together with some metabers of at least three different XP complementation groups, and it is, therefore, difficult to view the condition in the same light as TI'D (Lehmann and Norris, 1989), especially since all CS mutants are UV-sensitive and in some way repair-defectivc (Lehmann, 1982; Squires and Johnson, 1983; Venema et al., 1990). Mutations in many different genes thus appear to produce the clinical symptoms of CS and probably also the cellular defects, though when the mutation also generates an XP these v,iil probably be subsumed. Of likely importance for tumour aetiology is the finding that in these CS patients who do not also appear as XP, there is normal cellular immunity, a leature shared with repair defective patients with TI'D (Norris et al., 1988, 1990). Acknowledgements We are most grateful to our colleagues Drs. Colin Arlett, Dirk Bootsma, Francesco Giannelli, Jan Hoeijmakers, Brian Johnson, Alan Lehmann, Paul Norris, Mac Paterson, Alain Sarasin and Miria Stefanini for the gift of XPD fibroblasts or for information about particular patients, We are also indebted to Drs. David Mitchell and Rodney Nairn for use of data on CB (6-4) adduct re-

ill

moval, and to Stuart Linn for information on X P D and AP endonucleases and to Drs. C.A. W e b e r and L.H. Thompson for allowing us to add the note on the X P D correcting gene which appears below. We thank Jacquie Northfield and Peggy Pawley for excellent experimental help and the Cancer Research Campaign, of which R T J is a Research Fellow, for continued support.

Note added in proof

APPENDIXTABLE I COMMONXPD STRAIN SYNONYMS ATCC h

XplPO

NIGMS a identification GM 5424

XP5BE XP6BE

G M 10430 c

C R L 1157

XPI08LO XPIBR XPIKC

G M 0936 G M 3615 G M 1295

XP7BE

XPKABE XPINE XP2NE

identification CRL 1160 CRL 1200

CRL 1275 G M 0436 G M 0435

Dr. C.A. Weber and Dr. L.H. Thompson have presented evidence that the introduction of the human nucleotide excision repair gene ERCC2 specifically corrects the U V sensitivity of XP gr6up D cells, including the member previously designated XP group H, in a transient differential

XP3NE GM 0434 d XPI7BE GM 10428 xP-cs-2 e GM 3248 ~ NIGMS Human Genetic Mutant Cell Repository, Camden (NJ, U.S.A.). h American Type Culture Collection, Rockville(MD, U.S.A.).

cytotoxicity assay ( D N A Repair Workshop, Noordwijkerhout, The Netherlands, April 14-19, 1991).

c GM 8207 is the SV40 transformed version. d GM 10595 is the SV40 transformed version. ~ Also known as XP-SC patient 8 (Lafforet and Dupuy, 1978) and XP-SC-8 and XPH (MosheU et al., 1983a).

r XP58TO S LXP59TO XP17BE XPIDU XPI5PV ~-XP-CS-2 S L"Patient 7"

XPD/CS XPD/CS

0.65 a

0.7 e 0.38 a

0.47 a

XPIOTO XP43KO

0.4 a, 0.42 a 0.43 a 0.5 b 0.6 c

0.4 a

0.47 a

Do, U V ( J m - 2)

0.44 a 0.39 a

XP7BE

LXP6BE

Phenotype (if not strictly XPD)

XPI02LO XPI04LO [- XPI07LO S LXP108LO XPIIILO XP135LO XPIBR XPIKC XPKABE XPINE [" X P 2 N E S L XP3N1E

S

XPIPO XP5BE

Cell line

20 a

Ok 0k 15 '

0i

5-10 b 5-20 i

10 g

Cyclobutane dimer repair (% nonnaD

10 a

15 h

< 10 h

( 6 - 4 adduct) repair (% normal)

29

15

15 *

44

40-52

18-28

29-44

46-54 37-55

17-32

20

23--37

65-66 21-23

17 10 20

10 c

3.6 p

3.3 P, 6 q, 14 r, 2 s 3.3 p

paper m

v

20-30 I 30 2

41 c 25 a,x

25 z 35-55 d

near normal x 30-50 x; 32 y 30-50 x; 18; 25

9w

12 u 12 u 32u; 18

25--55 a

25-55 a; 10/27 t 25-55 a

data

Repair synthesis (UDS) % normal This Other

20-26

data

Host cell reactivation (% normal)

28

21

20

58 29

paper m

Incision % of normal This Other

R E P A I R CHARACTERISTICS O F S E L E C T E D X P D MI_rFANTS A N D X P D H E T E R O Z Y G O T E S

A P P E N D I X TABLE 2

present 3

present 5

absent 4

absent 4

absent 3 absent 4

AP endonuclease i

TTD Type 2

TFD Type 2

TI'D Type 3

TI'D Type 3

XPD heterozygote mother XPD heterozygote mother XPD heterozygote mother normal adult

normal embryo normal adult

embryonic lung

"I'FD IVT

TTDIBR

TI'DIBI

TTDIRO

XPHI02LO

BCL-D I

Hel

IBR

48BR

CRLI202

C R L ! 159

XPD/CS TI'D Type 2

XP8BR TI'D2GL

5f

5~

5 f

5 f

< I r

< 1f

< 1f

100. 8 Jm- 2 24h

100. 8 Jm - ". 24h f

8 Jm-2. 24 h f 50. 8 J m - 2. 24h f 0. 8 J m --~. 24h f 100. 8 Jm--'. 24h f 100, 8 Jm- 2 24h f

0.

1(110t

44 f

72 f

44 f

12 f

21 f

100 n IOO n

64

87

64

10

50 f

Of

0 f

IOO

1OO

1OO

20-21

< 10"

a Robbins ct al. (1974g Andrews et aL (1978). b Glover et al. (1979). c Johnson et at. (1985). d Ichihashi et at. (1988); Hiramoto et al. (1989);, (6-4) adducts, 40 Jm -2, 24 h. c Fujiwara and Salo (1985). f Broughton et at. (1990); 2 Jm -2, 30 min. . s Kantor and Hull (1984): 1 Jm -2, 20 day. h Mitchell and Nairn (1989): cyclobutane dime,s, !0 J i l l - " . 24 h~ (6-4) adducls, 10 Jm -2, 6 h. i Smerdon ct al. (1982); 12 Jm -2, 24 h. i 13. Mitchell, personal communication; 10 Jm -z, 24 h. x Paterson (1982); 15 Jm -2, 48 h. t Zelle and Lohman (1979): 2.9 Jm -2, 32 h. m This paper. For UDS cells U V irradiated in silo (20 J m - 2) and incubated with 5 / t C i / m l [3H]thymidine for I and 2 h. 100 nuclei scored per sample. For incision also see Figs. 1-3; 48BR taken as wild-t~A~e control. The incision data for all cells except XP3NE (4 Jm -2, 40 min*) are for 2 Jm -2, 40-min accumulations. " Squires et al. (1982); Squires and Johnson (1988). o Lehmann et al. (1988). P Day (1976). q Rainbow (i980). r Abrahams and van der Eb (1976). s Arase et at. (1979). t Kraemer et aL (1975). u Pawsey et ai. (1979), amniotic cells. v R.T. J o h n s o n , 10 Jm -2, I h post UV, fibroblasts. w Norris et at. (1990L x NIGMS Databoolc Y Kraemer et aL (1980). Takabe et aL (1977). t M. Stefanini, personal communication. 2 Moshell et ~1. (1983a). 3 S. Linn, personal communication. 4 Kuhnlein et al. (1978). 5 G.C. Elliott, R.T. Johnson and S. Linn, unpublished data.

Notes to Appendix table 2

4*

i!5 APPENDIX TABLE 3 SURVIVAL AFTER UV AND DNA EXCISION REPAIR IN XPD MUTANTS FROM THE MANNHEIM COLLECTION Cell line

D o Jm -2

Repair synthesis (% normal)

XP9MA XPI2MA a XPI7MA XPI8MA XPI9MA XP33MA XP36MA XP39MA XP40MA XP46MA XP47MA XP55MA

!.1 2.2 /5.11 1.4 !.0 0.84/1.6 1.0 0.84 0.80 0.62 3.35 1.24

22 100 76 75 48 28

Incision (% normal) 64 42 10 79 30

a 42-year-old sailor Data from Fischer et al. (1982); Thielmann et al. (1982, 1985).

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