Chromosoma(1992) 102:S17-$23
CHROMOSOMA 9 Springer-Verlag1992
Initiation of replication in the Chinese hamster dihydrofolate reductase domain Joyce L. Hamlin, Pieter A. Dijkwd, and James P. Vaughn Departmentof Biochemistryand CeUand MolecularBiologyProgram,Universityof VirginiaSchoolof Medicine, Charlottesville,VA 22908, USA ReceivedSeptember12, 1992
Abstract. Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ~50 kb "initiation locus" (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from an in vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiation per se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of 3Hthymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.
Abbreviations: BND-ceUulose,benzoylatednapthoylatedDEAEcel-
lulose; BrUdR,bromodeoxyuridine;CHO, Chinesehamsterovary; DHFR,dihydrofolatereductase;2-D,two-dimensional. Correspondence to: JoyceL. Hamlin
Introduction In order to facilitate the analysis of a defined chromosomal origin of replication, we have developed a methotrexate-resistant Chinese hamster ovary (CHO) cell line (CHOC 400) that has amplified the dihydrofolate reductase (DHFR) gene and flanking sequences ~1,000 times (Milbrandt et al. 1981). The major repeating unit (amplicon) in this cell line is 240 kb in length (Looney and Hamlin 1987; Ma et al. 1988), and the amplicons are arrayed as tandem clusters at three different chromosomal locations (Milbrandt et al. 1981). Employing in vivo labelling protocols on synchronized cells to pulse-label those amplified fragments that replicate first in the S period, we showed several years ago that initiation commences somewhere within a 28 kb region lying downstream from the DHFR gene (Heintz and Hamlin 1982; Heintz et al. 1983; Heintz and Hamlin 1983; contained within the bracketed region in Fig. I). Since then, we and others have devised several altemative strategies to attempt to localize initiation site(s) in this locus with greater resolution (Burhans et al. 1986; Anachkova and Hamlin 1989; Leu and Hamlin 1989; Handeli et al. 1989). One of these employed in-gel renaturation (Roninson 1983) to eliminate background labelling from single-copy sequences (Leu and Hamlin 1989). The resulting patterns suggested the presence of two separate initiation sites separated by about ~22 kb within the initiation locus (henceforth termed ori-I~ and ori-T) (Leu and Hamlin 1988). In more recent studies, we have analyzed replication intermediates in the amplified DHFR domain by two different 2-D gel electrophoretic mapping techniques (Brewer and Fangrnan 1987; Nawotka et al. 1988). The results of these studies led to the surprising conclusion
S18
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k~\x]
rillll
I
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k
~15 kb in length are synthesized in the proposed futile cycle, and presumably at every position within the ~50 kb initiation locus.
Conclusion As has been the case with most other areas o f eukaryotic biology, initiation o f replication in mammalian chromosomes appears to be more complicated than would have been predicted from simpler model systems. In addition, the characteristics o f the initiation mechanism in the D H F R locus appear to depend upon one's point o f view, i.e., the assay system. However, we suspect that these two complications are related: it is likely that each of the many assays that have been applied to this locus affords an accurate (though narrow) view o f the operative mechanism, but that we are unable to accommodate the data in a unifying model because key aspects o f the mechanism are unique to higher eukaryotic (or even mammalian) cells. Hopefully, new ways o f looking at this and other potential origins will help to clarify this presently cloudy field. Acknowledgements. We would like to thank our technicians, Carlton White and Kevin Cox, for expert assistancethroughout this project.We also thank the other members of out laboratory for helpful discussions and encouragement. This work was supported byNIH grants to J.L.H. and by an American Cancer Society award to P.A.D.
Anachkova B and Hamlin JL (1989) Replication in the amplified dihydro folate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol Cell Biol 9:532-540. Benbow RM (1985) Activation of DNA synthesis during early embryogenesis. Biology Fertil 3:299-345. Benbow RM, Gaudette MF, Hines PJ, Shioda M (1985) Initiation of DNA replication in eukaryotes. In: Cell Proliferation: Recent Advances (Boynton AL, Leffert HL, eds.), pp. 449-483 (Academic Press, New York). Brewer BJ Fangman WL (1987) The localization ofreplication origins on ARS plasmids in S. cerevisiae. Cell 51:463-471.
Burhans WC, Selegue JE, Heintz NH (1986) Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain. Proc Natl Acad Sci USA 83: 7790-7794. Burhans WC, Vassilev LT, Caddie MS, Heintz NH, DePamphilis ML (1990) Identification ofan origin of bidirectional DNA replication in mammalian chromosomes. Cell 62:955-965. Dijkwel PJ, Hamlin JL (1992) Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid, mimosine. In press, Mol Cell Bio112:3715-3722 Dijkwel PA, Vaugtm JP, Hamlin JL (1991) Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol Cell Biol 11:3850-3859. Handeli S, Klar A, Meuth M, Cedar H (1989) Mappingreplicationunits in animal cells. Cell 57:909-908. Heine NH, Hamlin JL (1982)An amplifiedchromosomalsequencethat includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci USA 79:4083-4087. Heintz NH, Hamlin JL (1983) In vivo effects of ara-C on DNA replicationin CHO ceils. II. Cytosine arabinoside effects the rate of DNA synthesis but not the pattern of replication of an am-plified chromosomal sequence at the onset of S. Biochemistry 22:3557. HeintzNI1,MilbrandtJD, GreisenKS, Hamlin JL (1983) Cloning of the initiation region of a mammalian chromosomal replicon. Nature 302:439--441. Lalande M (1990) A reversible arrest point in the late G 1phase of the mammalian cell cycle. Exp Cell Res 186:332-339. Leu TH, HamlinJL (1989) High resolution mapping ofreplication fork movementthrough the amplifieddihydrofolatereductasedomainin CHO cells by in-gel renaturation. Mol Cell Biol 9, 523-531. Levine AJ, Kang HS, Billheimer FE (1970) DNA replication in SV40infected cells. I. Analysis of replicating SV40 DNA. J Mol Biol 50:549-568. Linskens MHK, Huberman JA (1990) The two faces of higher eukaryotic DNA replication origins. Cell 62:845-847. Looney JE, Hamlin JL (1987) Isolation of the amplified dihydrofolate reductase domain from the methotrexate resistant CHO cells. Mol Cell Biol 7:569-577. Ma CA, Looney JE, Leu TH, Hamlin JL (1988) Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line. Mol Cell Biol 8:2316-2327. Milbrandt JD, Heintz NH, White WC, Rothman SM, Hamlin JL (1981) Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the gene for dihydrofolate reductase. Proc Natl Acad Sci USA78: 6043-6047 Mosca PJ, Dijkwel PA, Hamlin JL (1992) The plant amino acid, mimosine, inhibits initiation at a defined origin of replication in Chinese hamster cells. In press, Mol Cell Biol. Nawotka KA, Huberman, JA (1988) Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol 8, 1408-1413. Roninson I (1983) Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels. Nucleic Acids Res 14:5413-5431. Vaughn JP, Dijkwel PA, Hamlin JL (1990) Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075-1087.
CHROMOSOMA 9 Springer-Verlag1992
Initiation of replication in the Chinese hamster dihydrofolate reductase domain Joyce L. Hamlin, Pieter A. Dijkwd, and James P. Vaughn Departmentof Biochemistryand CeUand MolecularBiologyProgram,Universityof VirginiaSchoolof Medicine, Charlottesville,VA 22908, USA ReceivedSeptember12, 1992
Abstract. Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ~50 kb "initiation locus" (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from an in vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiation per se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of 3Hthymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.
Abbreviations: BND-ceUulose,benzoylatednapthoylatedDEAEcel-
lulose; BrUdR,bromodeoxyuridine;CHO, Chinesehamsterovary; DHFR,dihydrofolatereductase;2-D,two-dimensional. Correspondence to: JoyceL. Hamlin
Introduction In order to facilitate the analysis of a defined chromosomal origin of replication, we have developed a methotrexate-resistant Chinese hamster ovary (CHO) cell line (CHOC 400) that has amplified the dihydrofolate reductase (DHFR) gene and flanking sequences ~1,000 times (Milbrandt et al. 1981). The major repeating unit (amplicon) in this cell line is 240 kb in length (Looney and Hamlin 1987; Ma et al. 1988), and the amplicons are arrayed as tandem clusters at three different chromosomal locations (Milbrandt et al. 1981). Employing in vivo labelling protocols on synchronized cells to pulse-label those amplified fragments that replicate first in the S period, we showed several years ago that initiation commences somewhere within a 28 kb region lying downstream from the DHFR gene (Heintz and Hamlin 1982; Heintz et al. 1983; Heintz and Hamlin 1983; contained within the bracketed region in Fig. I). Since then, we and others have devised several altemative strategies to attempt to localize initiation site(s) in this locus with greater resolution (Burhans et al. 1986; Anachkova and Hamlin 1989; Leu and Hamlin 1989; Handeli et al. 1989). One of these employed in-gel renaturation (Roninson 1983) to eliminate background labelling from single-copy sequences (Leu and Hamlin 1989). The resulting patterns suggested the presence of two separate initiation sites separated by about ~22 kb within the initiation locus (henceforth termed ori-I~ and ori-T) (Leu and Hamlin 1988). In more recent studies, we have analyzed replication intermediates in the amplified DHFR domain by two different 2-D gel electrophoretic mapping techniques (Brewer and Fangrnan 1987; Nawotka et al. 1988). The results of these studies led to the surprising conclusion
S18
@ // I
il~])~"i
k~\x]
rillll
I
") k-
k
~15 kb in length are synthesized in the proposed futile cycle, and presumably at every position within the ~50 kb initiation locus.
Conclusion As has been the case with most other areas o f eukaryotic biology, initiation o f replication in mammalian chromosomes appears to be more complicated than would have been predicted from simpler model systems. In addition, the characteristics o f the initiation mechanism in the D H F R locus appear to depend upon one's point o f view, i.e., the assay system. However, we suspect that these two complications are related: it is likely that each of the many assays that have been applied to this locus affords an accurate (though narrow) view o f the operative mechanism, but that we are unable to accommodate the data in a unifying model because key aspects o f the mechanism are unique to higher eukaryotic (or even mammalian) cells. Hopefully, new ways o f looking at this and other potential origins will help to clarify this presently cloudy field. Acknowledgements. We would like to thank our technicians, Carlton White and Kevin Cox, for expert assistancethroughout this project.We also thank the other members of out laboratory for helpful discussions and encouragement. This work was supported byNIH grants to J.L.H. and by an American Cancer Society award to P.A.D.
Anachkova B and Hamlin JL (1989) Replication in the amplified dihydro folate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol Cell Biol 9:532-540. Benbow RM (1985) Activation of DNA synthesis during early embryogenesis. Biology Fertil 3:299-345. Benbow RM, Gaudette MF, Hines PJ, Shioda M (1985) Initiation of DNA replication in eukaryotes. In: Cell Proliferation: Recent Advances (Boynton AL, Leffert HL, eds.), pp. 449-483 (Academic Press, New York). Brewer BJ Fangman WL (1987) The localization ofreplication origins on ARS plasmids in S. cerevisiae. Cell 51:463-471.
Burhans WC, Selegue JE, Heintz NH (1986) Isolation of the origin of replication associated with the amplified Chinese hamster dihydrofolate reductase domain. Proc Natl Acad Sci USA 83: 7790-7794. Burhans WC, Vassilev LT, Caddie MS, Heintz NH, DePamphilis ML (1990) Identification ofan origin of bidirectional DNA replication in mammalian chromosomes. Cell 62:955-965. Dijkwel PJ, Hamlin JL (1992) Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid, mimosine. In press, Mol Cell Bio112:3715-3722 Dijkwel PA, Vaugtm JP, Hamlin JL (1991) Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol Cell Biol 11:3850-3859. Handeli S, Klar A, Meuth M, Cedar H (1989) Mappingreplicationunits in animal cells. Cell 57:909-908. Heine NH, Hamlin JL (1982)An amplifiedchromosomalsequencethat includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci USA 79:4083-4087. Heintz NH, Hamlin JL (1983) In vivo effects of ara-C on DNA replicationin CHO ceils. II. Cytosine arabinoside effects the rate of DNA synthesis but not the pattern of replication of an am-plified chromosomal sequence at the onset of S. Biochemistry 22:3557. HeintzNI1,MilbrandtJD, GreisenKS, Hamlin JL (1983) Cloning of the initiation region of a mammalian chromosomal replicon. Nature 302:439--441. Lalande M (1990) A reversible arrest point in the late G 1phase of the mammalian cell cycle. Exp Cell Res 186:332-339. Leu TH, HamlinJL (1989) High resolution mapping ofreplication fork movementthrough the amplifieddihydrofolatereductasedomainin CHO cells by in-gel renaturation. Mol Cell Biol 9, 523-531. Levine AJ, Kang HS, Billheimer FE (1970) DNA replication in SV40infected cells. I. Analysis of replicating SV40 DNA. J Mol Biol 50:549-568. Linskens MHK, Huberman JA (1990) The two faces of higher eukaryotic DNA replication origins. Cell 62:845-847. Looney JE, Hamlin JL (1987) Isolation of the amplified dihydrofolate reductase domain from the methotrexate resistant CHO cells. Mol Cell Biol 7:569-577. Ma CA, Looney JE, Leu TH, Hamlin JL (1988) Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line. Mol Cell Biol 8:2316-2327. Milbrandt JD, Heintz NH, White WC, Rothman SM, Hamlin JL (1981) Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the gene for dihydrofolate reductase. Proc Natl Acad Sci USA78: 6043-6047 Mosca PJ, Dijkwel PA, Hamlin JL (1992) The plant amino acid, mimosine, inhibits initiation at a defined origin of replication in Chinese hamster cells. In press, Mol Cell Biol. Nawotka KA, Huberman, JA (1988) Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol 8, 1408-1413. Roninson I (1983) Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels. Nucleic Acids Res 14:5413-5431. Vaughn JP, Dijkwel PA, Hamlin JL (1990) Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075-1087.
