Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

Mutation Research, 174 (1986) 303-308

303

Elsevier

MRLett. 0885

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19 M . J . Siciliano a, A . V . C a r r a n o b a n d L . H . T h o m p s o n h aDepartment of Genetics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030 and bBiomedical Sciences Division L-452, Lawrence Livermore National Laboratory, University of California, Livermore, CA 94550 (U.S.A.) (Accepted 4 April 1986)

Summary The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinesehamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repairdeficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.

Following exposure to mutagens and isolation of clones whose growth was retarded by subsequent exposure to low levels of far ultraviolet light (UVL), over 140 DNA-repair-deficient, Chinese hamster ovary (CHO) cell lines were isolated (Thompson and Carrano, 1983). Representative mutants were shown to have no UVL-induced repair replication because they fail to perform the nicking step of nucleotide excisiOn repair (Thompson et al., 1983b). Therefore, the cells resemble the phenotype of the human disease xeroderma pigmentosum (XP) - - a DNA-repair deficiency with a high frequency of skin cancer (Cleaver, 1983). Also, like XP cells, the CHO excision-repair

mutants fail to repair damage from large-adduct chemicals, and appear, from complementation tests, to result from mutations in a series of genes associated with the repair process. The CHO mutants fall into 5 complementation groups (Thompson et al., 1981). Another class of CHOrepair deficiency mutation (exemplified by the line EM9) was obtained by its hypersensitivity to the alkylating agent, ethyl methanesulfonate (Thompson et al., 1982a). This phenotype has a spectrum of agent sensitivity and other characteristics that are very different from the excision-repairdeficient mutants (Thompson et al., 1985a). The response of EM9 to UVI is near normal, but it is

0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

304 hypersensitive to ionizing radiation and defective in rejoining strand breaks in DNA. Like the cells of another human DNA-repair syndrome with a high frequency of cancer, Bloom syndrome (Chaganti et al., 1974), EM9 has an extremely high rate of sister-chromatid exchange (SCE) in untreated cultures. However Bloom syndrome cells do not show a defect in repairing strand breaks after alkylation damage or ionizing radiation. Their biochemical defect may be different from that of EM9. By somatic cell genetic methods a human gene, excision-repair complementing defective repair in Chinese hamster 1 (ERCC1), which corrected the excision-repair deficiency in the CHO mutant UV20 of hamster UVL complementation Group 2, was shown to be on human chromosome 19 (Thompson et al., 1985b). This assignment was confirmed by molecular hybridization, to members of a human-rodent hybrid clone panel, of a human DNA probe associated with correction of the same CHO deficiency (Rubin et al., 1985). Here we identify the human chromosome carrying the gene complementing the SCEinducing mutation present in CHO EM9.

onto nitrocellulose filters (Southern, 1975), to the p7f12 probe for chromosome 13 (Cavenee et al., 1984) and the sis oncogene for chromosome 22 (Dalla Favara et al., 1981). Because widespread breakage and rearrangement of human chromosomes in hybrids with this fusion and selection scheme had taken place, detailed direct karyotype analysis by a combination of Giemsa-11 staining and reverse banding with chromomycin/methylgreen (Thompson et al., 1985c) was used on only certain selected hybrids. Subclones that had lost resistance to CldUrd were obtained by culturing hybrid clones for periods of from weeks to months in normal medium, subcloning into 96 well trays, and making a duplicate culture of each subclone. Duplicate sets were incubated in selective medium to determine which subclones of the original set were sensitive to CldUrd (killed in 4-5 days). C e r t a i n CldUrd-resistant and sensitive hybrid clones and subclones were analysed for SCE frequency as previously described (Thompson et al., 1982) by growing for two cycles in medium containing l0/~M BrdUrd. After 25 h, colcemid was added and slides for microscope analysis of SCEs were prepared.

Materials and methods

Culture conditions of EM9 cells as well as the methods used to PEG-hydridize them to human lymphocytes or fibroblasts were as those we used for UV20 cells and their hybridization (Thompson et al., 1985c). 24 h after fusion, hybrids were then selected in~CldUrd medium (8/~M CldUrd, 32 #M dThd, 10 #M FdUrd, 200/~M dCyd) which kills EM9 cells but not normal CHO cells (Thompson et al., 1985b). Isozyme analysis to detect the presence of all human chromosomes in hybrid cells except the 13 and 22 (markers for these chromosomes were not available in this fusion scheme) was conducted as before (Thompson et al., 1985c). Presences of human chromosomes 13 and 22 were determined in hybrid clones by hybridization of the clone's appropriately digested DNAs, which had been electrophoresed onto agarose gels and blotted

Results and discussion

11 independent, CldUrd-resistant, hybrid clones were produced from the lymphocyte fusion (prefixed 9HL) and one hybrid was obtained from the fibroblast fusion (9HF3). The results of the analysis of human chromosomes present in the hybrids are presented in Table 1. All 12 CldUrdresistant clones are true hybrids as indicated by the presence of at least one marker representing a human chromosome in each. Material from only one human chromosome (No. 19) was consistently present in the CldUrd-resistant hybrids. An isozyme marker for human chromosome 19, GPI, was present in 11 of th 12 hybrid clones (92o7o). Markers for any other human chromosome were only infrequently present D in only 2007o of the hybrids on average, with no other human

305

TABLE

1

MARKERS HYBRID

FOR HUMAN

CHROMOSOMES

PRESENT

(+) AND ABSENT

(-)

IN CIdUrd-RESISTANT

CHO

EM9

x HUMAN

CLONES

Hybrid

Markers

clones

1

for human

chromosomes

2

3

4

5

+

+

+

+

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

+

+

+

+

+

+

+

-

+

+

-

+

22

X

+

9HL1 3

+

+

+

5

+

6 +

7

+

8

+

9

+

10

+

11

+

12

+

13

.

-

.

+

.

.

-

+

+

-

+

+

+

-

+

+

.

.

9HF3

.

.

+

-

+

-

+

-

+

+

+

+

+

+

+

+

.

+

+

+

+

ND a-

+ +

+

-

+

+

+

-

+

-

ND

+

+

ND

+ +

30

92

ND

+

-

_

_

17

8

42

17

+ +

ND

-

ND

-

0

8

% with marker

for

chromosome

17

8

25

17

25

17

17

33

30

25

8

33

8

25

33

aND, not done.

chromosome present in more than 42°7o of the hybrid clones. Hybrid clones with human GPI had as much human as Chinese hamster form of the isozyme, suggesting that the marker was present in almost every cell. This uniformity was verified in clone 9HL10, which shows the typical pattern of generally equal amounts of human and hamster GPI (as seen in human GPI-containing clones 9HL12 and -13 and 9HF3 in Fig. 1) and for which cytogenetic analysis revealed the presence of at least one human chromosome 19 in 22 of 22 cells examined. This is the expected result for a human chromosome complementing a genetic effect in hybrid cells maintained under selection. Although no human chromosome 19 material could be identified cytogenetically or biochemically in the exceptional CldUrd-resistant clone, 9HL1, the presence of sequences for the human 19 could not be excluded since G-11 banding indicated unidentifiable human chromosomal material (data not shown). The phenomenon of breakage and partial loss and/or translocation of human chromosomal material was not unique to clone 9HL1 in this fusion scheme. For instance, we were similarly

unable to cytogenetically observe human chromosome 19 material in clone 9HL5, yet, the GPI marker is as well represented there as in clone 9HL10. However, the second human isozyme marker for chromosome 19, PEPD, was absent in 9HL5 (as in 9HF3, Fig. 1). Therefore the discordancy of 9HL1 with respect to its CldUrdresistance and lack of observable human chromosome 19 marker is likely due to chromosome breakage in hybrid cells (Siciliano et al., 1984). The lack of human PEPD in 9HL5 and 9HF3, and its presence in quantitatively lesser amounts than the hamster form of the isozyme (as in 9HL13 of Fig. 1) in 8 of the 9 independent hybrids with PEPD, indicate that the human gene complementing EM9 may be closer to GP1 than PEPD on chromosome 19. To further test the role of human chromosome 19 material to correct the EM9 phenotype, certain CldUrd-resistant hybrid clones were subcloned to produce sensitive subclones and/or sensitive subclones and resistant subclones. The clones used in these experiments and their derived subclones are listed in Table 2 along with their status with

306

t

5--5

12

9HL I 13 1 0 - 8 0

t

H

C

3

9HF J 3-31

TABLE 2 P R E S E N C E ( + ) A N D ABSENCE ( - ) OF H U M A N C H R O M O S O M E 19 M A R K E R (GPI) A N D SCE F R E Q U E N CY IN P R I M A R Y CldUrd-RESISTANT HYBRID CLONES A N D T H E I R RESISTANT A N D SENSITIVE SUBCLONES

CC

HH S

R

R

S

R

S

HH

Hybrid clones and subclones

CldUrdresistant (R) or sensitive (S)

Human GPI

SCEs/ chromosomea

9HLI 9HLI-1 9HLI-I1

R R S

-

0.72 + 0.05 0.37 _+ 0.03 5.45 + 0.21

9HL5 9HL5-5

R S

+ -

0.80 ± 0.09 4.26 +_ 0.14

9HLI0 9HLI0-80

R S

+ -

0.60 ± 0.04 5.02 + 0.19

9HF3 9HF3-50 9HF3-31

R R S

+ + -

0.29 ± 0.02 0.51 + 0.04 3.70 ± 0.14

CC

Fig. I. Z y m o g r a m s o f GPI (top panel) and P E P D (bottom panel) showing the electrophoretic forms o f these enzymes in certain 9HL and 9 H F hybrid clones and subclones as well as h u m a n HeLa (H) and C H O (C) control samples. Channels containing the different samples are marked across the top. CldUrd resistance (R) or sensitivity (S) is indicated for each hybrid sample between the two panels. Origins (O) o f each of the gels are indicated in the right margin. Anodal ends are toward the top and cathodal ends toward the bottom o f each panel. Each o f the enzymes are dimeric in structure and the positions of the h u m a n (HH) and Chinese hamster (CC) homodimers for each enzyme are indicated in the left margin. Since C H O c h r o m o s o m e s are not lost from the hybrid cells, all hybrids have the hamster homodimer for each enzyme. This is the only band present in hybrids where the h u m a n gene has segregated. Hybrids retaining the h u m a n gene for an enzyme will produce a heterodimeric band intermediate in electrophoretic mobility between the h u m a n and Chinese hamster homodimers. In hybrid clones where the h u m a n gene for the enzyme is retained in almost all of the cells, there are generally equal a m o u n t s o f h u m a n and hamster gene products producing a relatively symmetrical 3-banded pattern with greatest activity in the middle of heterodimeric band (e.g., GPI in 9 H L I 2 , 13 and 9HF3; P E P D in 9HLI2). Where there has been some segregation of the h u m a n gene for an enzyme a m o n g cells of a hybrid clone which have the gene, the isozyme pattern will be skewed in favor of the hamster homodimer so that there will be more activity in that band than in the heterodimeI (e.g., P E P D in 9HL13).

"SCEs were scored on 25 metaphases. Since the n u m b e r of chromosomes per cell varied both within and a m o n g the hybrid clones, the SCE frequencies are given on a per c h r o m o s o m e basis, rather than on a per cell basis.

respect to CldUrd-resistance, presence of the human GPI, and their observed SCE frequencies. With the exception of subclones derived from 9HL1, the return of CldUrd-sensitivity correlated perfectly with the loss of human GPI (Fig. 1). Clones and subclones retaining the human GPI not only retained CldUrd-resistance but also had SCE frequencies that were not appreciably different than have been reported for normal CHO cells, i.e., 0.47 per chromosome (Thompson et al., 1985b). In the hybrid subclones which segregated human GPI (9HL5-5, 9HL10-80, and 9HF3-31), SCE frequencies increased dramatically to approach those seen in EM9 cells, i.e., 5.60 per chromosome (Table 2). From these accumulated data, therefore, we conclude that the human gene complementing the repair deficiency present in EM9, which we designate RCC for repair complementing defective repair in Chinese hamster, is located on chromosome 19 - - closer to GPI than

307

PEPD. Hybrid clones with discordancies between PEPD, GPI and RCC should prove extremely valuable in analysis with a series of human chromosome 19 molecular markers to regionally assign RCC. Thus human chromosome 19 corrects the recessive defects in both EM9 and UV20. A priori, the fact that the phenotypes of these two mutants are distinctly different would not exclude the possibility that the same gene might be involved in each mutation. However, complementation tests have shown that hybrids formed between EM9 and UV20 are resistant to both CldUrd and UVL (our unpublished results). This two-way correction of defects indicates that the two phenotypes arise from the deficiencies at different genetic loci. There are several significant aspects to our finding that the first two human repair genes assigned by their correction of CHO mutations are located on chromosome 19. We have shown (Siciliano et al., 1983) that the isozyme genes on the human 19, GPI and PEPD, are also syntenic in the Chinese hamster (on the hamster chromosome 9), as they are in all mammalian species where they have been assigned as well as in amphibians (D.A. Wright et al., 1983) and fish (J.E. Wright et al., 1983). Our findings that chromosome 9 is hemizygous in CHO cells (Siciliano et al., 1983) and the high frequency, approximately 10 -3 , at which recessive repair mutations can be induced at these two loci in CHO cells (Thompson et al., 1980) suggest that these repair genes are likely also to be on the hamster chromosome 9 as part of a highly conserved region of the vertebrate genome. These results underscore the value of rodent cell lines, containing various hemizygous regions, for isolating recessive mutations. With this approach complex phenotypes, such as DNA-repair deficiencies, can be readily attributed to multiple genes dispersed throughout the human karyotype. Subsequent molecular cloning of different human genes identified in this way may then aid in determining the exact biochemical defect(s) underlying important mutations such as the EM9. Finally, the results suggest that the identification of polymorphic human gene probes in regions where clusters of repair genes are so assign-

ed would appear to be a practical enterprise. Such probes would then be the tools in studies on the inheritance of cancer in XP, Bloom syndrome, and other cancer families where repair deficiency as a factor in carcinogenesis is a reasonable hypothesis.

Acknowledgements We are greatly appreciative of the excellent technical support of B. White, P. Morris, K. Burkhart-Schultz, J. Minkler, S. Stewart and C. Mooney in various phases of this work. We also acknowledge the support of the NIH (CA34797, CA04484 and CA34936), the'Exxon Corporation, and the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation, and a gift from Kenneth D. Muller. A portion of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract No. W-7405-ENG-48.

References Cavenee, W., R. Leach, T. Mohandas, P. Pearson and R. White (1984) Isolation and regional localization of DNA segments revealing polymorphic loci from human chromosome 13, Am. J. Hum. Genet., 36, 10-24. Chaganti, R.S.K., S. Schonberg and J. German (1974) A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. (U.S.A.), 71, 4508-4512. it. Cleaver, J.E. 0983)Xeroderma pigmentosum, in: J.R. Stanbury, J.B. Wyngaarden, D.S. Frederickson, J.L. Goldstein and M.S. Brown (Eds.), The Molecular Basis of Inherited Disease,. McGraw-Hill, New York, pp. 1227-1248. Dalla Favera, R., E.P. Gelmann, R.C. Gallo and F. WongStaal (1981) A human onc gene homologous to the transforming gene (v-sis) of simian sarcoma virus, Nature (London), 292, 31-35. Rubin, J.S., V.R. Prideaux, H.P. Willard, A.M. Dulhanty, G.F. Whitmore and A. Bernstein (1985) Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene, Mol. Cell Biol., 5,398-405. Siciliano, M.J., R.L. Stallings, G.M. Adair, R.M. Humphrey and J. Siciliano 0983) Provisional assignment of TPI, G P I and P E P D to Chinese hamster autosomes 8 and 9: a cytogenetic basis for functional haploidy of an autosomal linkage group in CHO cells, Cytogenet. Cell Genet., 35, 15-20.

308 Siciliano, M.J., R.E.K. Fournier and R.L. Stallings (1984) Regional assignment of A D A and ITPA to mouse chromosome 2 (Cl-ter), J. Heredity, 75, 175-180. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98, 503-517. Thompson, L.H., J.S. Rubin, J.E. Cleaver, G.F. Whitmore and K. Brookman (1980) A screening method for isolating DNA repair-deficient mutants of CHO cells, Somat. Cell Genet., 6, 391-405. Thompson, L.H., D.B. Busch, K. Brookman, C.L. Mooney and D.A. Glaser (1981) Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells, Proc. Natl. Acad. Sci. (U.S.A.), 78, 3734-3737. Thompson, L.H., K.W. Brookman, L.E. Dillehay, A.V. Carrano, J.A. Mazrimas, C.L. Mooney and J.L. Minkler (1982a) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister chromatid exchange, Mutation Res., 95, 427-440. Thompson, L.H., K.W. Brookman, L.E. Dillehay, C.L. Mooney and A.V. Carrano (1982b) Hypersensitivity to mutation and sister-chromatid exchange induction in CHO cell mutants defective in incising DNA containing UV lesions, Somat. Cell Genet., 8, 759-773. Thompson, L.H., and A.V. Carrano (1983) Analysis of mammalian cell mutagenesis and DNA repair using in vitro selected CHO cell mutants, in: E.C. Freidberg and B.A. Bridges (Eds.), U.C.L.A. Symposia on Molecular and Cellular Biology, N.S. Vol. 11, Liss, New York, pp. 125-143.

Thompson, L.H., K.W. Brookman and C.L. Mooney (1984) Repair of DNA adducts in asynchronous CHO cells and the role of repair in cell killing and mutation induction in synchronous cells treated with 7-bromo-methylbenz[a]anthracene, Somat. Cell Mol. Genet., 10, 183-194. Thompson, L.H., K.W. Brookman, J.L. Minkler, J.C. Fuscoe, K.A. Henning and A.V. Carrano (1985a) DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange, Mol. Cell Biol. 5, 881-884. Thompson, L.H., C.L. Mooney, K. Burkhart-Schultz, A.V. Carrano and M.J. Siciliano (1985b) Correction of a nucleotide-excision-repair mutation by human chromosome 19 in hamster-human hybrid cells, Somat. Cell Mol. Genet., 11, 87-92. Wright, D.A., C.M. Richards, J.S. Frost, A.M. Camozzi and R.J. Kunz (1983a) Genetic mapping in amphibians, in: M.C. Rattazzi, J.G. Scandalios and G.S. Whitt (Eds.), Isozymes: Current Topics in Biological and Medical Research, Vol. 10, Liss, New York, pp. 287-311. Wright, J.E., K. Johnson, A. Hollister and B. May (1983b) Meiotic models to explain classical linkage, pseudolinkage, and chromosome pairing in tetraploid derivative salmonid genomes, in: M.C. Rattazzi, J.G. Scandalios and G.S. Whitt (Eds.), Isozymes: Current Topics in Biological and Medical Research, Vol. 10, Liss, New York, pp. 239-260. Communicated by R.J. Preston