Nucleic Acids Research, Vol. 19, No. 12 3455
©) 1991 Oxford University Press
Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems Werner Pansegrau and Erich Lanka* Max-Planck-Institut fur Molekulare Genetik, Abteilung Schuster, Ihnestrasse 73, D-1000 Berlin 33, FRG Submitted April 29, 1991 Site and strand specific nicking at a transfer origin (ori7) is the central event in initiation of transfer DNA replication during bacterial conjugation. Recently, sequence identity in a DNA region adjacent to the nick sites of IncP plasmids RP4 and R751 and part of the border sequences of agrobacterial Ti and Ri plasmids has been demonstrated (Figure 1 and ref. 1). A search for this 'nick region' in a variety of transfer origins of other plasmids demonstrates its conservation in R64 (IncII) and pTFFC2, a mobilizable plasmid from Thiobacillus ferrooxidans (Figure 1, ref. 2). This finding is paralleled by occurrence of a common sequence motif in proteins (DNA-relaxases) encoded by the respective plasmids that interact with their cognate nick regions (Figure 2). Using the corresponding consensus sequence, a search in protein data bases (NBRF-Protein Release 27.0; Swiss-Prot Release 16.0) revealed that the 'relaxase motif is also occurring in potential DNA-relaxing proteins (Rlx) of mobilizable plasmids from S.aureus (Figure 2).
RK2/RP4
TGGGCC T ACTTC A C C T A T C C T TTAGCTAACTTC A C A CAT C CT
R751
R64 pTF-FC2 pTiT37 Nop (LB) pTIT37 Nop (RB)
pTIA pTIA
G6TATTACAATT GCACATCCTGTC G T 6TGTTACAAC 66TCATCCT6TA 6 T T T AC AC C AC A ATATATCCTGCC G T T T AC CC6C CA ATATATCC TGTC 6TTTACACCACA A TAT AT C CT GVC C 6TTTACCC6CCA A T A T A T C C T G'T C ATTTACAATT6A A TAT A T C C T'G C C AATTACAACGGT A T A T A T C C T'G C C ATTTACAATT6A ATATATCCT6CC AATTACAACGGT A TA TAT CC TGC C T T T T AC AA T T 6A ATATATCCTGCC AATTACAAC6GT ATATATCCTGCC TTTTACACCTCG A T A T A T c c T GCC AA T T AC AA C CG C A T TATCCTGCC
Nop (L) Nop (RB) Oct (LBa) Oct (RBb) Oct (U a) Oct (RBb) Oct (LB8a) Oct (fSb)
pTAcH5 pTIACH5 pT115955 pT115955 pT115655 Oct (1 c) pT115655 Oct (RBd) pRM (LB a) (RSb) PRA4 (U c) pRM (RBd)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Waters et al. (1991) Proc. Natl. Acad. Sci. USA 88, 1456-1460. Rawlings et al. (1984) J. Bacteriol. 158, 737-738. Pansegrau et al. (1988) Biochim. Biophys. Acta 951, 365-374. Furuya et al. (1991) J. Bacteriol. 173, 2231-2237. Rohrer et al. (1991) J. Bacteriol. 173, in press. Jouanin et al. (1989) Plant Mol. Biol. 12, 75-85. Durrenberger et al. (1989) Proc. Natl. Acad. Sci. USA 86, 9154-9158. Albright et al. (1987) J. Bacteriol. 169, 1046-1055. Gielen et al. (1984) EMBO J. 3, 835-846. Ziegelin et al. (1991) DNA Sequence 1, 303-327. Jajaswal et al. (1987) J. Bacteriol. 169, 5035-5045. Hirajama et al. (1988) MoI. Gen. Genet. 213, 229-237. Brenner and Shaw (1985) EMBO J. 4, 561-568. Projan et al. (1988) Nucd. Acids Res. 16, 2179-2187. Dreher and Matsura (1988) unpublished.
* To
whom correspondence should be addressed
A
(4) (5) (6)
A
A A A A GA A A A
(6) (8) (8) (6) (6) (9) (9) (6) (6) (6 (6)
(6) (6) (6)
CCTGACCACAAG ATATATCCTGTC ATA T cc CT ATCCTG GGCC TA
ACKNOWLEDGEMENTS
REFERENCES
(3) (3)
A A GTTTACATCACA ATATATCCTGCC A CATGACAG6AAC ATATATCCTGTC A TTTTACGTTGGC ATATATCCTGCC
Cw-
We are grateful to Heinz Schuster for generous support. We thank Douglas E. Rawlings and Jurg Rohrer for providing the pTFFC2 sequence data and Barbara Hohn for helpful suggestions. The work was financially supported by the Deutsche Forschungsgemeinschaft grant SFB 344/B2.
GVC C
GvC C
Figure 1. Alignment of defined and potential nick regions of various plasmids. Conserved nucleotide positions are boxed. Locations of nick-sites are indicated by wedges. References are given in brackets.
Tral Tral NikB MobA VirD2 1irD2 Rix Rlx Rlx
(RP4) (R751) (R64) (pTF-FC2) (pTiA6) (pRiA4) (pC221) (pSl 94) (pC223)
Consensus
104 R 104 R 153 Y 51 A 126 Y 126 Y
V I V I L L 94 V A 94 V A 94 V A a
S A SA S A Y G T A T A V Y V Y V Y
V V V L Y F T T T
H H H H H H H H H H
H N T A V I T T N
D D D D D D D D D
D
T T T T R R K K T
D D D D D D D D D
N N N N H H H H H
D HN
L L L L P P Y Y V
H H H H H H H H H
H
I I V L L L N N N
H H H H H H H H H
I I V L V V I I I
A A A A V V V I V
I I V I V V I I I
N K N K N IIIR N IIIR N IIIR N R N S N S N S
I I V V R R V V I
H H H H E E
P P P P L L D L N L D L
T T E E L L E E E
(10) (10) (4) (5) (11) (12) (13) (14) (15)
H a a a N
Figure 2. Alignment of DNA-relaxases. Numbers indicate the sequence positions of the first amino acid residues shown. A common motif is boxed. References are given in brackets.
©) 1991 Oxford University Press
Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems Werner Pansegrau and Erich Lanka* Max-Planck-Institut fur Molekulare Genetik, Abteilung Schuster, Ihnestrasse 73, D-1000 Berlin 33, FRG Submitted April 29, 1991 Site and strand specific nicking at a transfer origin (ori7) is the central event in initiation of transfer DNA replication during bacterial conjugation. Recently, sequence identity in a DNA region adjacent to the nick sites of IncP plasmids RP4 and R751 and part of the border sequences of agrobacterial Ti and Ri plasmids has been demonstrated (Figure 1 and ref. 1). A search for this 'nick region' in a variety of transfer origins of other plasmids demonstrates its conservation in R64 (IncII) and pTFFC2, a mobilizable plasmid from Thiobacillus ferrooxidans (Figure 1, ref. 2). This finding is paralleled by occurrence of a common sequence motif in proteins (DNA-relaxases) encoded by the respective plasmids that interact with their cognate nick regions (Figure 2). Using the corresponding consensus sequence, a search in protein data bases (NBRF-Protein Release 27.0; Swiss-Prot Release 16.0) revealed that the 'relaxase motif is also occurring in potential DNA-relaxing proteins (Rlx) of mobilizable plasmids from S.aureus (Figure 2).
RK2/RP4
TGGGCC T ACTTC A C C T A T C C T TTAGCTAACTTC A C A CAT C CT
R751
R64 pTF-FC2 pTiT37 Nop (LB) pTIT37 Nop (RB)
pTIA pTIA
G6TATTACAATT GCACATCCTGTC G T 6TGTTACAAC 66TCATCCT6TA 6 T T T AC AC C AC A ATATATCCTGCC G T T T AC CC6C CA ATATATCC TGTC 6TTTACACCACA A TAT AT C CT GVC C 6TTTACCC6CCA A T A T A T C C T G'T C ATTTACAATT6A A TAT A T C C T'G C C AATTACAACGGT A T A T A T C C T'G C C ATTTACAATT6A ATATATCCT6CC AATTACAACGGT A TA TAT CC TGC C T T T T AC AA T T 6A ATATATCCTGCC AATTACAAC6GT ATATATCCTGCC TTTTACACCTCG A T A T A T c c T GCC AA T T AC AA C CG C A T TATCCTGCC
Nop (L) Nop (RB) Oct (LBa) Oct (RBb) Oct (U a) Oct (RBb) Oct (LB8a) Oct (fSb)
pTAcH5 pTIACH5 pT115955 pT115955 pT115655 Oct (1 c) pT115655 Oct (RBd) pRM (LB a) (RSb) PRA4 (U c) pRM (RBd)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Waters et al. (1991) Proc. Natl. Acad. Sci. USA 88, 1456-1460. Rawlings et al. (1984) J. Bacteriol. 158, 737-738. Pansegrau et al. (1988) Biochim. Biophys. Acta 951, 365-374. Furuya et al. (1991) J. Bacteriol. 173, 2231-2237. Rohrer et al. (1991) J. Bacteriol. 173, in press. Jouanin et al. (1989) Plant Mol. Biol. 12, 75-85. Durrenberger et al. (1989) Proc. Natl. Acad. Sci. USA 86, 9154-9158. Albright et al. (1987) J. Bacteriol. 169, 1046-1055. Gielen et al. (1984) EMBO J. 3, 835-846. Ziegelin et al. (1991) DNA Sequence 1, 303-327. Jajaswal et al. (1987) J. Bacteriol. 169, 5035-5045. Hirajama et al. (1988) MoI. Gen. Genet. 213, 229-237. Brenner and Shaw (1985) EMBO J. 4, 561-568. Projan et al. (1988) Nucd. Acids Res. 16, 2179-2187. Dreher and Matsura (1988) unpublished.
* To
whom correspondence should be addressed
A
(4) (5) (6)
A
A A A A GA A A A
(6) (8) (8) (6) (6) (9) (9) (6) (6) (6 (6)
(6) (6) (6)
CCTGACCACAAG ATATATCCTGTC ATA T cc CT ATCCTG GGCC TA
ACKNOWLEDGEMENTS
REFERENCES
(3) (3)
A A GTTTACATCACA ATATATCCTGCC A CATGACAG6AAC ATATATCCTGTC A TTTTACGTTGGC ATATATCCTGCC
Cw-
We are grateful to Heinz Schuster for generous support. We thank Douglas E. Rawlings and Jurg Rohrer for providing the pTFFC2 sequence data and Barbara Hohn for helpful suggestions. The work was financially supported by the Deutsche Forschungsgemeinschaft grant SFB 344/B2.
GVC C
GvC C
Figure 1. Alignment of defined and potential nick regions of various plasmids. Conserved nucleotide positions are boxed. Locations of nick-sites are indicated by wedges. References are given in brackets.
Tral Tral NikB MobA VirD2 1irD2 Rix Rlx Rlx
(RP4) (R751) (R64) (pTF-FC2) (pTiA6) (pRiA4) (pC221) (pSl 94) (pC223)
Consensus
104 R 104 R 153 Y 51 A 126 Y 126 Y
V I V I L L 94 V A 94 V A 94 V A a
S A SA S A Y G T A T A V Y V Y V Y
V V V L Y F T T T
H H H H H H H H H H
H N T A V I T T N
D D D D D D D D D
D
T T T T R R K K T
D D D D D D D D D
N N N N H H H H H
D HN
L L L L P P Y Y V
H H H H H H H H H
H
I I V L L L N N N
H H H H H H H H H
I I V L V V I I I
A A A A V V V I V
I I V I V V I I I
N K N K N IIIR N IIIR N IIIR N R N S N S N S
I I V V R R V V I
H H H H E E
P P P P L L D L N L D L
T T E E L L E E E
(10) (10) (4) (5) (11) (12) (13) (14) (15)
H a a a N
Figure 2. Alignment of DNA-relaxases. Numbers indicate the sequence positions of the first amino acid residues shown. A common motif is boxed. References are given in brackets.
