V o l u m e 266, n u m b e r 1,2, 115-117
FEBS 08517
J u n e 1990
Isolation and characterization of a human c D N A clone encoding a novel D N A topoisomerase II homologue from HeLa cells Caroline A. Austin and L. Mark Fisher Molecular Genetics Group, Department o f Cellular and Molecular Sciences, St George's Hospital Medical School, University o f London, London, UK Received 26 M a r c h 1990; revised version received 27 April 1990 We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial eDNA clones from a HeLa carcinoma cell eDNA library. Two clones were identical to an internal fragment of HeLa topo II eDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II eDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologne is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines. DNA topoisomerase II; Human cell line; eDNA cloning; Iscenzyme; Protein sequence homology
1. INTRODUCTION DNA topoisomerase II (EC 5.99.1.3; topo II) is an important nuclear scaffold protein implicated in key biological processes including DNA replication and chromosome segregation [1-3]. The enzyme is a homodimer that acts to pass a duplex DNA segment through a transient enzyme-bridged double strand break in DNA [3,4]. Several clinically useful anticancer agents, e.g. etoposide and doxorubicin, appear to exert their cytotoxic effects by interfering with this enzymatic DNA breakage-reunion reaction [5]. Human topo II was first purified as a 170 kDa polypeptide (p170) from the HeLa cervical carcinoma cell line for which Wang and colleagues reported the cDNA sequence, and we in parallel obtained a homologous partial eDNA clone (CAA1) [6,7]. However, very recent work has demonstrated the presence in the Burkitt lymphoma cell line Raji of two isoenzyme forms of topo II: IIo~, analogous to p170 in HeLa cells, and a 180 kDa form, termed Ilfl or plB0 [8]. Analysis of partial cDNA clones isolated from a Raji library indicated that these proteins derive from, two different mRNA species which are differentially expressed in the cell cycle [9]. During the course of eDNA cloning studies using CAA1 as a hybridization probe, we isolated a novel human HeLa cDNA clone that encodes an as yet Correspondence address: C.A. Austin, Dept of Cellular and Molecular Sciences, Division of Biochemistry, St George's Hospital Medical School, Cranmer Terrace, London SWI7 ORE, UK
unreported topo II-related polypeptide. In light of current interest in different forms of topo II and their biological roles, we describe here the initial characterization of the novel eDNA and its relationship to cDNAs for the human p170 and plB0 topo II isoenzymes.
2. EXPERIMENTAL 2.1. cDNA cloning A human HeLa cell ~,gtl0 eDNA library was obtained from Dr D. Gewert. It was constructed by oligo dT priming of mRNA (isolated from interferon-treated HeLa ceils) and the resulting cDNAs were cloned into )~gt10 using EcoRI linkers. The library was screened under conditions of low stringency with a partial eDNA clone (CAAI) encoding part of the C-terminal region of the piT0 form of human topoisomerase I1 [7]. CAA1 was isolated by cross-species expression screening of a HepG2 )~gtll library and spans a 231 base pair (bp) region between nucleotide positions 3495 and 3726 of the 4753bp eDNA sequence [6,7]. Hybridisation was carried out at 42°C in buffer containing 6 x SSC, 5 x Denhardts, 0.5070 sodium dodecyl sulphate, 100/~g/ml denatured salmon sperm DNA and 8°70 dextran sulphate. Positive clones were plaque purified by at least 3 successive rounds of screening. Phage )~DNA was isolated as described [10]. 2.2. DNA sequence analysis Insert DNA from ~,gtl0 clones released by digestion with EcoRI, was purified by eiectrophoresis in a 2070 low gelling agarose gel (Sigma) and isolated on NA45 paper (Schleicher and Schuell). Insert DNA or fragments arising from digestion with HaelII or BgllI were ligated into appropriately cut M 13mpl0 and mpl 1 RF DNA and used to transform E. coli XL1 recA. Single stranded recombinant MI3 DNA was purified and sequenced by the Sanger dideoxy method using either Klenow fragment (Amersham) or Sequenase II (United States Biochemical Corp.). Sequence information was analysed using the Genofit/PC Gene Package (Inteiligenetics).
Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies
115
Volume 266, number 1,2
FEBS LETTERS
3. RESULTS
June 1990
A EcoRl
EcoRl
3.1. Isolation o f cDNA clones homologous to human p170 DNA topoisomerase H Screening of a HeLa ~,gtl0 cDNA library with the partial p170 topo II cDNA CAA1 yielded 3 positive clones, two of which had an identical 1.7 kb EcoRI insert (CAA4) while the third contained a 700 bp insert (CAA5) (Fig. 1). The restriction map of CAA4 was identical to part of p170 topo II cDNA (Fig. 1) and this identification was confirmed by DNA sequence analysis. CAA4 spans nucleotide positions 2014-3698 of p170 topo II cDNA encoding amino acid residues 660-1220 of the 1530 residue protein. The probe CAA1 is therefore identical to the 3' end of clone CAA4 (Fig.
1).
I
Es
Haelll
Bglll
295
423
20
TT C~AT~A F F D
7(]2
40
CG A T T A A G T T A T T A C G G T ~ A C G T A A G G A G T G G C T ~ T G G G A A T G T T G L R L S ¥ ¥ G L R K E W L V G M L
I00
80
GGAGCAGAATCTACAAAGCTTAACAATCAAGCCCGTTTCATTTTAGAGAAGATACAAGGG G A E S T K L N N O A R F I L E K I Q 140
AAAATTACTATAG AGAATAGGTCAAAGAAAGATTTGATTCAAATGTTAGTCCAGAGA
The second type of cDNA clone, CAA5, isolated at the same time under the same conditions as CAA4, had a different restriction map to those of CAA4 and p170 cDNA (Fig. 1) and suggested it may encode a homologous but novel form of DNA topoisomerase II.
3.2. Protein encoded by the novel human cDNA CAA5 aligns and shares substantial homology with human p170 topo H The 702 bp nucleotide sequence of CAA5 was determined and found to exhibit 62% homology with a segment of the p170 topo II cDNA - nucleotide positions 3062-3764 (Figs 1, 2). CAA5 has a single uninterrupted open reading frame encoding a 234 amino acid residue polypeptide (Fig. 2B). The CAA5 protein sequence aligns with residues 1021-1253 of p170 topo II displaying a 70% overall homology (58% identical residues and 12% conserved residues) (Fig. 3). Homology with
K
I
T
l
E
N
R
S
K
K
D
L
I
HEHH H
H B
IIII I I I
H H HH
E
I I II
I
1
L
V
Q
GGT
R
G
260
280
GAAACACAAAACCAGCATGATGATAGTTCCTCCGATTCAGGAACTCCTTCAGGCCCAGAT E T Q N Q H D D S S S D S G T P S G P
320
D
340
TTTAATTATATTTTAAATATGTCTCTG TGGTCTCTTA CTAAAGAAAAAGTTGAAGAA CTG F N Y I L N M S L W S L T K E K V E E L 380
400
ATTAAAC AGAGAGATG CAAAAG GG CGA GAGG T CAATGATCTTAAAAGAAAATCTCCTTCA I K Q R D A K G R E V N D L K R K S P S
440 G ATCT~GGAAAGAGGATTTAG D L W K E D L
460 CGG CATTTG TTGAAGAACTGGATAAAGTGGAATCTCAA A A F V E E L D K V E S Q
500
520
G AACGAGAAGATGTTCTGGCTGGAATGTCTGGAAAAGCAATTAAAGGTAAAGTTGG E R E D V L A G M S G K A I K G K V
560
G
CAAA K
580
C CTAAG GTGAAGAAACT CCAG TTGG AAGAG A CAATG CC CTCACCTTATGGCAGAAGAATA P K V K K L Q L E E T M P S P Y G R R I
i"
640
CTATG AAGGCAGATGCCAG CA~GTTG A M K A D A S K K 680
/
4753
M
TATGAAT CTGACCCAGTG AAAG CCTGGAAAGAAG CACAAGAAAAGG CAGCAGAAGAGGAT Y E S D P V K A W K E A Q E K A A E E D
620
i
Q 220
200
A TT CCTGA~TTACAG I P E I T
p170
G
160
GGTGATC~GATACTGCAGCAGTA~GTGGAAT~GATGAA G D L D T A A V K V E F D
L
CTG AAGAAGAAGAAG L K K K K
700
E
L._J 250bp
EH E
CAM
LJ 349~ E
3726 E
Fig. 2. D N A sequence analysis of CAA5, a topo II-related HeLa cDNA. (A) D N A sequencing strategy. (B) Nucleotide sequence of CAA5 and the derived amino acid sequence of its single open reading frame.
Ii 3898
2014 E
cAA5
HB
I
E
1 702
E
plS0
[" 1
BH BH
tl
HH
H B
H
II
I I I
FFD~I~Y-YG [ / ~ K E W L V G M L G A E S T K ~ N Q A R F I LEKIQGKI T I E N R S K K D L I Q M L V Q R G LFELRLKYYG L R K E W L L G M L G A E S A K L N N Q A R F I LEKI DGKI I I ENKPKKELI KVLI QRG
61
YES DPVKAWKEAQEI~EAEEDE- - -TQNQHDDSS S D S G T P S G P D F N Y I L N M S LMS LTKEKV
118
EELI K Q R D A K G R E V N D L I ~ K S PS D L W K E D L A A F V E E L D K V E S Q E R E D V L A G M S G K A I KGK
178
V G K P K V K K L Q L E E T M P S PYGRRI I PE I T - A M K A D A S K K L L K K ~ G D L D T A A V K V E F D E
E
] 2685
Fig. 1. Restriction maps and alignment of topo II cDNA clones, pl70 and p180 isoenzyme maps were generated from D N A sequence in references [6] and [9]. E, H and B denote restriction sites for EcoRI, HaeIII and BglII. CAA1 and CAA4 are identical with p170 c D N A between the indicated nucleotide positions. CAA5 and p 180 are aligned by their respective homology with the p170 cDNA sequence.
116
1 1021
1201
G ..... ~ T Q ~ E V L P S
PRGQRVI PRI T I E M ~ E A E K K N K K K I A E N T E G S P Q E ~ V E
Fig. 3. Alignment of the predicted amino acid sequence of CAA5 (top line) with that of p170 topo II [6] showing identical (:) and conserved residues (-). Amino acid residues conserved between CAA5 and Drosophila topo II [11] are overlined.
Volume 266, number 1,2
FEBS LETTERS
p170 topo II is especially marked at the N-terminal end of the CAA5 polypeptide: of the first 81 residues, 62 are identical and a further 10 are conserved substitutions. Significant homology at the protein level is also observed between CAA5 and the appropriate region of topo II from other species including Drosophila (Fig. 3) and yeast (not shown). Comparison of CAA5 with the recently published partial cDNA and protein sequence of the human p 180 topo II isoenzyme [9] (Fig. 1) did not reveal significant homology (not shown). 4. DISCUSSION Topo II from HeLa cells has previously been isolated only as a 170 kDa polypeptide form [4]. The corresponding cDNA has been described and the suggestion made that HeLa topo II is encoded by a single copy gene, mapped to chromosome 17 [6]. Our cDNA cloning studies reported here indicate that HeLa cells also express another protein, partially encoded by CAA5, that exhibits remarkable sequence conservation with p170 topo II and other eukaryotic topo II enzymes and indeed may represent a distinct human isoenzyme form. At present, we do not know whether the CAA5-encoded protein is the same as or different from the p180 topo II isoenzyme described in Raji cells [8]. The longest partial cDNA clone (SPll) for p180 isolated from a nitrogen mustard resistant Raji line, appears to encode the N-terminal region of the protein, judged by its homology to nucleotide region 379-3065 of the p170 topo II cDNA (Fig. 1) [5,9]. In contrast, CAA5 encodes protein sequence homologous to the Cterminal end of p170 topo II and does not overlap that determined for p180 (Fig. 1). It is interesting, however, that the p180 and CAA5 sequences are homologous to directly adjacent regions of p170 topo II, each sequence terminating at an EcoRI site in the respective cDNA (Fig. 1). The derived amino acid sequence for p180 topo II reported by Chung et al. [9] finishes at a position equivalent to residue 1021 of the p170 form (terminating in the cDNA at a natural EcoRI site) while that from CAA5 is homologous to p170 residues 1021-1253 (Fig. 1). Thus, the p180 and CAA5 partial cDNA clones could correspond to contiguous EcoRI cDNA fragments. Conceivably, CAA5 encodes a C-terminal segment of the p180 topo II isoenzyme. Chung et al. isolated their Raji p180 topo II partial cDNA clone by screening with a probe encoding Drosophila topo II [9]. In contrast, CAA5 was obtained
June 1990
using a C-terminal human HeLa topo II probe (Fig. 1). It is known that HeLa, Drosophila and other eukaryotic topo II cDNA sequences are closely homologous in the N-terminal two thirds of the coding sequence whereas nucleotide sequences for the Cterminal regions are more divergent [11]. These considerations may explain why the different screening approaches yielded topo II-related cDNA clones that align with different ends of the HeLa p170 cDNA sequence. Topo II p170 and p180 isoenzymes have been detected in cell extracts from a number of sources in addition to Raji cells including human U937 monocyte and COLO 201 colon carcinoma cell lines and in murine P388 leukaemia and NIH 3T3 fibroblast cell lines [9]. HeLa p170 topo II was isolated before the existence of isoenzyme forms was generally recognized. It will now be important to examine HeLa cell extracts for the presence of other topo II proteins, e.g. p180. The physiological and chemotherapeutic roles of topo II isoenzymes are of great interest and remain to be elucidated. Further work is in progress to characterise fully the structure, expression and biological function of the novel CAA5 encoded HeLa protein. Acknowledgements: We thank Dirk Gewert for the HeLa kgtl0 cDNA library and Edward Margerrison and Mike Clemens for discussion. This work was supported by the Cancer Research Campaign and by the Gunnar Nilsson Cancer Research Trust Fund.
REFERENCES [1] Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. [2] Wang, J.C. (1987) Biochim. Biophys. Acta 909, 1-9. [3] Vosberg, H.-P. (1985) Curr. Top. Microbiol. Immunol. 114, 19-102. [4] Miller, K.G., Liu, L.F. and England, P.T. (1981) J. Biol. Chem. 256, 9334-9339. [5] Rose, K.M. (1988) FASEB J. 2, 2474-2478. [6] Tsai-Pflugfelder, M., Liu, L.F., Liu, A.A., Tewey, K.M., Whang-Peng, J., Knutsen, T., Huedner, K., Croce, C.M. and Wang, J.C. (1988) Proc. Natl. Acad. Sci. USA 85, 7177-7181. [7] Austin, C.A., Barot, H., Margerrison, E.E.C., Hayes, M.V. and Fisher, L.M. (1989) Biochem. Soc. Trans. 17, 528-529. [8] Drake, F.H., Hofmann, G.A., Bartus, H.F., Mattern, M.R., Crooke, S.T. and Mirabelli, C.K. (1989) Biochemistry 28, 8154-8160. [9] Chung, T.D.Y., Drake, F.H., Tan, K.B., Per, S.R., Crooke, S.T. and Mirabelli, C.K. (1989) Proc. Natl. Acad. Sci. USA 86, 9431-9435. [10] Austin, C.A. (1986) PhD Thesis, University of London. [11] Wykcoff, E., Natalie, D., Nolan, J.M., Lee, M. and Hsieh, T.S. (1989) J. Mol. Biol. 205, 1-13.
117
FEBS 08517
J u n e 1990
Isolation and characterization of a human c D N A clone encoding a novel D N A topoisomerase II homologue from HeLa cells Caroline A. Austin and L. Mark Fisher Molecular Genetics Group, Department o f Cellular and Molecular Sciences, St George's Hospital Medical School, University o f London, London, UK Received 26 M a r c h 1990; revised version received 27 April 1990 We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial eDNA clones from a HeLa carcinoma cell eDNA library. Two clones were identical to an internal fragment of HeLa topo II eDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II eDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologne is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines. DNA topoisomerase II; Human cell line; eDNA cloning; Iscenzyme; Protein sequence homology
1. INTRODUCTION DNA topoisomerase II (EC 5.99.1.3; topo II) is an important nuclear scaffold protein implicated in key biological processes including DNA replication and chromosome segregation [1-3]. The enzyme is a homodimer that acts to pass a duplex DNA segment through a transient enzyme-bridged double strand break in DNA [3,4]. Several clinically useful anticancer agents, e.g. etoposide and doxorubicin, appear to exert their cytotoxic effects by interfering with this enzymatic DNA breakage-reunion reaction [5]. Human topo II was first purified as a 170 kDa polypeptide (p170) from the HeLa cervical carcinoma cell line for which Wang and colleagues reported the cDNA sequence, and we in parallel obtained a homologous partial eDNA clone (CAA1) [6,7]. However, very recent work has demonstrated the presence in the Burkitt lymphoma cell line Raji of two isoenzyme forms of topo II: IIo~, analogous to p170 in HeLa cells, and a 180 kDa form, termed Ilfl or plB0 [8]. Analysis of partial cDNA clones isolated from a Raji library indicated that these proteins derive from, two different mRNA species which are differentially expressed in the cell cycle [9]. During the course of eDNA cloning studies using CAA1 as a hybridization probe, we isolated a novel human HeLa cDNA clone that encodes an as yet Correspondence address: C.A. Austin, Dept of Cellular and Molecular Sciences, Division of Biochemistry, St George's Hospital Medical School, Cranmer Terrace, London SWI7 ORE, UK
unreported topo II-related polypeptide. In light of current interest in different forms of topo II and their biological roles, we describe here the initial characterization of the novel eDNA and its relationship to cDNAs for the human p170 and plB0 topo II isoenzymes.
2. EXPERIMENTAL 2.1. cDNA cloning A human HeLa cell ~,gtl0 eDNA library was obtained from Dr D. Gewert. It was constructed by oligo dT priming of mRNA (isolated from interferon-treated HeLa ceils) and the resulting cDNAs were cloned into )~gt10 using EcoRI linkers. The library was screened under conditions of low stringency with a partial eDNA clone (CAAI) encoding part of the C-terminal region of the piT0 form of human topoisomerase I1 [7]. CAA1 was isolated by cross-species expression screening of a HepG2 )~gtll library and spans a 231 base pair (bp) region between nucleotide positions 3495 and 3726 of the 4753bp eDNA sequence [6,7]. Hybridisation was carried out at 42°C in buffer containing 6 x SSC, 5 x Denhardts, 0.5070 sodium dodecyl sulphate, 100/~g/ml denatured salmon sperm DNA and 8°70 dextran sulphate. Positive clones were plaque purified by at least 3 successive rounds of screening. Phage )~DNA was isolated as described [10]. 2.2. DNA sequence analysis Insert DNA from ~,gtl0 clones released by digestion with EcoRI, was purified by eiectrophoresis in a 2070 low gelling agarose gel (Sigma) and isolated on NA45 paper (Schleicher and Schuell). Insert DNA or fragments arising from digestion with HaelII or BgllI were ligated into appropriately cut M 13mpl0 and mpl 1 RF DNA and used to transform E. coli XL1 recA. Single stranded recombinant MI3 DNA was purified and sequenced by the Sanger dideoxy method using either Klenow fragment (Amersham) or Sequenase II (United States Biochemical Corp.). Sequence information was analysed using the Genofit/PC Gene Package (Inteiligenetics).
Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies
115
Volume 266, number 1,2
FEBS LETTERS
3. RESULTS
June 1990
A EcoRl
EcoRl
3.1. Isolation o f cDNA clones homologous to human p170 DNA topoisomerase H Screening of a HeLa ~,gtl0 cDNA library with the partial p170 topo II cDNA CAA1 yielded 3 positive clones, two of which had an identical 1.7 kb EcoRI insert (CAA4) while the third contained a 700 bp insert (CAA5) (Fig. 1). The restriction map of CAA4 was identical to part of p170 topo II cDNA (Fig. 1) and this identification was confirmed by DNA sequence analysis. CAA4 spans nucleotide positions 2014-3698 of p170 topo II cDNA encoding amino acid residues 660-1220 of the 1530 residue protein. The probe CAA1 is therefore identical to the 3' end of clone CAA4 (Fig.
1).
I
Es
Haelll
Bglll
295
423
20
TT C~AT~A F F D
7(]2
40
CG A T T A A G T T A T T A C G G T ~ A C G T A A G G A G T G G C T ~ T G G G A A T G T T G L R L S ¥ ¥ G L R K E W L V G M L
I00
80
GGAGCAGAATCTACAAAGCTTAACAATCAAGCCCGTTTCATTTTAGAGAAGATACAAGGG G A E S T K L N N O A R F I L E K I Q 140
AAAATTACTATAG AGAATAGGTCAAAGAAAGATTTGATTCAAATGTTAGTCCAGAGA
The second type of cDNA clone, CAA5, isolated at the same time under the same conditions as CAA4, had a different restriction map to those of CAA4 and p170 cDNA (Fig. 1) and suggested it may encode a homologous but novel form of DNA topoisomerase II.
3.2. Protein encoded by the novel human cDNA CAA5 aligns and shares substantial homology with human p170 topo H The 702 bp nucleotide sequence of CAA5 was determined and found to exhibit 62% homology with a segment of the p170 topo II cDNA - nucleotide positions 3062-3764 (Figs 1, 2). CAA5 has a single uninterrupted open reading frame encoding a 234 amino acid residue polypeptide (Fig. 2B). The CAA5 protein sequence aligns with residues 1021-1253 of p170 topo II displaying a 70% overall homology (58% identical residues and 12% conserved residues) (Fig. 3). Homology with
K
I
T
l
E
N
R
S
K
K
D
L
I
HEHH H
H B
IIII I I I
H H HH
E
I I II
I
1
L
V
Q
GGT
R
G
260
280
GAAACACAAAACCAGCATGATGATAGTTCCTCCGATTCAGGAACTCCTTCAGGCCCAGAT E T Q N Q H D D S S S D S G T P S G P
320
D
340
TTTAATTATATTTTAAATATGTCTCTG TGGTCTCTTA CTAAAGAAAAAGTTGAAGAA CTG F N Y I L N M S L W S L T K E K V E E L 380
400
ATTAAAC AGAGAGATG CAAAAG GG CGA GAGG T CAATGATCTTAAAAGAAAATCTCCTTCA I K Q R D A K G R E V N D L K R K S P S
440 G ATCT~GGAAAGAGGATTTAG D L W K E D L
460 CGG CATTTG TTGAAGAACTGGATAAAGTGGAATCTCAA A A F V E E L D K V E S Q
500
520
G AACGAGAAGATGTTCTGGCTGGAATGTCTGGAAAAGCAATTAAAGGTAAAGTTGG E R E D V L A G M S G K A I K G K V
560
G
CAAA K
580
C CTAAG GTGAAGAAACT CCAG TTGG AAGAG A CAATG CC CTCACCTTATGGCAGAAGAATA P K V K K L Q L E E T M P S P Y G R R I
i"
640
CTATG AAGGCAGATGCCAG CA~GTTG A M K A D A S K K 680
/
4753
M
TATGAAT CTGACCCAGTG AAAG CCTGGAAAGAAG CACAAGAAAAGG CAGCAGAAGAGGAT Y E S D P V K A W K E A Q E K A A E E D
620
i
Q 220
200
A TT CCTGA~TTACAG I P E I T
p170
G
160
GGTGATC~GATACTGCAGCAGTA~GTGGAAT~GATGAA G D L D T A A V K V E F D
L
CTG AAGAAGAAGAAG L K K K K
700
E
L._J 250bp
EH E
CAM
LJ 349~ E
3726 E
Fig. 2. D N A sequence analysis of CAA5, a topo II-related HeLa cDNA. (A) D N A sequencing strategy. (B) Nucleotide sequence of CAA5 and the derived amino acid sequence of its single open reading frame.
Ii 3898
2014 E
cAA5
HB
I
E
1 702
E
plS0
[" 1
BH BH
tl
HH
H B
H
II
I I I
FFD~I~Y-YG [ / ~ K E W L V G M L G A E S T K ~ N Q A R F I LEKIQGKI T I E N R S K K D L I Q M L V Q R G LFELRLKYYG L R K E W L L G M L G A E S A K L N N Q A R F I LEKI DGKI I I ENKPKKELI KVLI QRG
61
YES DPVKAWKEAQEI~EAEEDE- - -TQNQHDDSS S D S G T P S G P D F N Y I L N M S LMS LTKEKV
118
EELI K Q R D A K G R E V N D L I ~ K S PS D L W K E D L A A F V E E L D K V E S Q E R E D V L A G M S G K A I KGK
178
V G K P K V K K L Q L E E T M P S PYGRRI I PE I T - A M K A D A S K K L L K K ~ G D L D T A A V K V E F D E
E
] 2685
Fig. 1. Restriction maps and alignment of topo II cDNA clones, pl70 and p180 isoenzyme maps were generated from D N A sequence in references [6] and [9]. E, H and B denote restriction sites for EcoRI, HaeIII and BglII. CAA1 and CAA4 are identical with p170 c D N A between the indicated nucleotide positions. CAA5 and p 180 are aligned by their respective homology with the p170 cDNA sequence.
116
1 1021
1201
G ..... ~ T Q ~ E V L P S
PRGQRVI PRI T I E M ~ E A E K K N K K K I A E N T E G S P Q E ~ V E
Fig. 3. Alignment of the predicted amino acid sequence of CAA5 (top line) with that of p170 topo II [6] showing identical (:) and conserved residues (-). Amino acid residues conserved between CAA5 and Drosophila topo II [11] are overlined.
Volume 266, number 1,2
FEBS LETTERS
p170 topo II is especially marked at the N-terminal end of the CAA5 polypeptide: of the first 81 residues, 62 are identical and a further 10 are conserved substitutions. Significant homology at the protein level is also observed between CAA5 and the appropriate region of topo II from other species including Drosophila (Fig. 3) and yeast (not shown). Comparison of CAA5 with the recently published partial cDNA and protein sequence of the human p 180 topo II isoenzyme [9] (Fig. 1) did not reveal significant homology (not shown). 4. DISCUSSION Topo II from HeLa cells has previously been isolated only as a 170 kDa polypeptide form [4]. The corresponding cDNA has been described and the suggestion made that HeLa topo II is encoded by a single copy gene, mapped to chromosome 17 [6]. Our cDNA cloning studies reported here indicate that HeLa cells also express another protein, partially encoded by CAA5, that exhibits remarkable sequence conservation with p170 topo II and other eukaryotic topo II enzymes and indeed may represent a distinct human isoenzyme form. At present, we do not know whether the CAA5-encoded protein is the same as or different from the p180 topo II isoenzyme described in Raji cells [8]. The longest partial cDNA clone (SPll) for p180 isolated from a nitrogen mustard resistant Raji line, appears to encode the N-terminal region of the protein, judged by its homology to nucleotide region 379-3065 of the p170 topo II cDNA (Fig. 1) [5,9]. In contrast, CAA5 encodes protein sequence homologous to the Cterminal end of p170 topo II and does not overlap that determined for p180 (Fig. 1). It is interesting, however, that the p180 and CAA5 sequences are homologous to directly adjacent regions of p170 topo II, each sequence terminating at an EcoRI site in the respective cDNA (Fig. 1). The derived amino acid sequence for p180 topo II reported by Chung et al. [9] finishes at a position equivalent to residue 1021 of the p170 form (terminating in the cDNA at a natural EcoRI site) while that from CAA5 is homologous to p170 residues 1021-1253 (Fig. 1). Thus, the p180 and CAA5 partial cDNA clones could correspond to contiguous EcoRI cDNA fragments. Conceivably, CAA5 encodes a C-terminal segment of the p180 topo II isoenzyme. Chung et al. isolated their Raji p180 topo II partial cDNA clone by screening with a probe encoding Drosophila topo II [9]. In contrast, CAA5 was obtained
June 1990
using a C-terminal human HeLa topo II probe (Fig. 1). It is known that HeLa, Drosophila and other eukaryotic topo II cDNA sequences are closely homologous in the N-terminal two thirds of the coding sequence whereas nucleotide sequences for the Cterminal regions are more divergent [11]. These considerations may explain why the different screening approaches yielded topo II-related cDNA clones that align with different ends of the HeLa p170 cDNA sequence. Topo II p170 and p180 isoenzymes have been detected in cell extracts from a number of sources in addition to Raji cells including human U937 monocyte and COLO 201 colon carcinoma cell lines and in murine P388 leukaemia and NIH 3T3 fibroblast cell lines [9]. HeLa p170 topo II was isolated before the existence of isoenzyme forms was generally recognized. It will now be important to examine HeLa cell extracts for the presence of other topo II proteins, e.g. p180. The physiological and chemotherapeutic roles of topo II isoenzymes are of great interest and remain to be elucidated. Further work is in progress to characterise fully the structure, expression and biological function of the novel CAA5 encoded HeLa protein. Acknowledgements: We thank Dirk Gewert for the HeLa kgtl0 cDNA library and Edward Margerrison and Mike Clemens for discussion. This work was supported by the Cancer Research Campaign and by the Gunnar Nilsson Cancer Research Trust Fund.
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