k.=) 1991 Oxford University Press
`
`Nucleic Acids Research, Vol. 19, No. 15 4045-4057
`
`Compilation and alignment of DNA polymerase sequences
`
`Junetsu Ito and Dan K.Braithwaite
`Department of Microbiology and Immunology, College of Medicine, University of Arizona Health
`Sciences Center, Tucson, AZ 85724, USA
`
`Received May 29, 1991; Revised and Accepted July 9, 1991
`
`INTRODUCTION
`More than 40 different DNA polymerases, including some
`putative DNA polymerase sequences deduced from nucleotide
`sequence data, have recently been reported (1-39). The amino
`acid sequences of these DNA polymerases have been aligned and
`partial homologous regions identified by many investigators
`(2-4,9,10,12-25,27-36,42-51). Based on the segmental
`amino acid sequence similarities, DNA polymerases have been
`classified into two major groups; E. coli DNA polymerase I-Type
`and eukaryotic DNA polymerase ca-Type (14,44,47,48,51), or
`family A DNA polymerases and family B DNA polymerases
`(4,9,50). As the number of DNA polymerase sequences
`increases, the classification of DNA polymerases becomes
`increasingly ambiguous. For example, DNA polymerase delta
`of yeast was shown to have amino acid sequence similarity to
`the a-Type DNA polymerases (17). It has become necessary to
`establish a unified classification of DNA polymerases. Here we
`propose to classify DNA polymerases into families A, B, and
`C (Figure 1: A, B, and C), according to the amino acid sequence
`II, and III,
`homologies with E. coli DNA polymerases I,
`respectively. As new and different prokaryotic and eukaryotic
`DNA polymerases are identified, the number of families can
`easily be expanded by using additional letters of the alphabet (i.e.,
`D, E, etc.).
`The bacterium E. coli (strain K12) contains three distinct DNA
`polymerases I, II, and HI (52). E. coli DNA polymerase I, the
`first DNA polymerase discovered, is specified by the polA gene
`(52). E. coli DNA polymerase II, encoded by the polB gene,
`was recently sequenced and found to be identical to the dinA
`gene, a DNA damage inducible gene whose expression is
`regulated by the SOS system in E. coli (8,53). Amino acid
`sequence alignment shows that E. coli DNA polymerase II has
`significant homology with family B (a-Type) DNA polymerases
`(8,53,54).
`E. coli DNA polymerase IH is a multisubunit enzyme encoded
`by various dna genes (55); the DNA polymerizing a-subunit
`encoded by the polC (dnaE) gene (56) and the 3'- 5' exonuclease
`performing e-subunit encoded by the dnaQ gene (57). The a-
`subunit of E. coli DNA polymerase III exhibits an extensive
`homology with the corresponding a-subunit of Salmonella
`typhimurium DNA polymerase m (35); and both show significant
`homology to Bacillus subtilis DNA polymerase HI, a single-
`polypeptide encoded by the polC gene (36).
`In summary, family A DNA polymerases are named for their
`homology to the product of the polA gene encoding E. coli DNA
`polymerase I; family B DNA polymerases are named for their
`
`homology to the product of the polB gene encoding E. coli DNA
`polymerase 11; and family C DNA polymerases are named for
`their homology to the product of the polC gene encoding E. coli
`DNA polymerase HI.
`The eukaryotic DNA polymerase (3, the smallest known DNA
`polymerase, does not have homology with those of any of the
`DNA polymerase families described above. Instead, DNA
`polymerase (3 has homology with terminal transferases (37). This
`,B group we will call family X (Figure ID). The classification
`and original reference(s) for the amino acid sequences of each
`DNA polymerase are shown in Table 1.
`All of the family A DNA polymerases, except for yeast
`mitochondrial DNA polymerase I, are prokaryotic and are very
`sensitive to dideoxynucleotide inhibitors, and therefore are useful
`enzymes for DNA sequencing by the chain-termination method
`(58). The family A DNA polymerases are resistant to aphidicolin.
`The family B DNA polymerases are quite extensive in number
`and variety. Most of the family B DNA polymerases, if not all,
`aphidicolin and relatively
`sensitive
`to
`resistant
`to
`are
`dideoxynucleotide inhibitors. Most of the family B DNA
`polymerases, except for pAI2 (33) and yeast DNA polymerase
`II (16), contain the highly conserved amino acid sequence motif
`YGDTD, which has been suggested to form part of the dNTP
`binding site. Amino acid substitutions in this conserved sequence
`resulted in defects in the DNA polymerase activity without
`affecting the 3'- 5' exonuclease activity (59,60,61). The family
`C DNA polymerases are major bacterial replicative DNA
`polymerases which do not have appreciable homology with those
`of family A and B DNA polymerases. B.
`subtilis DNA
`polymerase Im is a single polypeptide that is highly sensitive to
`hydroxyphenylazouracil (62). It is anticipated that the number
`of sequenced family C DNA polymerases will increase rapidly,
`since all of the aerobic bacteria may contain a member of this
`family of DNA polymerases.
`
`SEQUENCE ALIGNMENT
`The 37 complete DNA polymerase sequences and 3 complete
`terminal deoxynucleotidyltransferase (TDT) sequences are listed
`in 4 groups; the family A DNA polymerases, the family B DNA
`polymerases, the family C DNA polymerases, and family X DNA
`polymerases (including TDTs). In order to limit the space needed
`for the alignment, we omitted DNA polymerase sequences that
`are very similar to the prototype DNA polymerase. The DNA
`polymerases not shown include: herpes virus type-2 (63),
`
`Illumina Ex. 1116
`IPR Petition - USP 10,435,742
`
`

`

`4046 Nucleic Acids Research, Vol. 19, No. 15
`
`adenovius Wpe-S (64), bacteriophage T3 (65), and bacteriophage
`PZA (66).
`
`ACCURACY OF SEQUENCE DATA
`Whenever a sequence ambiguity existed in a published sequence,
`we contacted the authors to obtain the updated sequence
`a few publisd amino acid sequences
`information. We found
`differ at one or more positions from their GenBank/EMBL entry.
`Again, we have communicated with the primary author to confirm
`the correct sequences.
`The multiple alignment of the amino acid sequences was
`obained by a series of pairwise aligments combined and adjusted
`by eye into larger and larger subsets of similar sequences. The
`process of combining and adjusting by eye was aided by modified
`versions of the MOTIF program (67) and the ALIGN program
`(68). The GAP and BESTFIT programs, from UWGCG
`(University of Wisconsin Genetic Computer Group) (69), initay
`generated the pairwise alignments, adjusted for maximum
`alignment that allowed for a considerable number of gaps. We
`then compressed these alignments by eye to give a more
`contiguous alignment. The alignment of the sequences for optimal
`similarity is straightforward in the areas of relatively conserved
`strucure, but is much more arbitary in the more varied sequence
`areas. The alignment of the varied areas should therefore be
`regarded as less than optimal in view of the difficulties concerned
`with multiple alignments in these areas.
`Finally, we invite fiuther correction from readers, and welcome
`suggested revisions and altemative alignments.
`
`ACKNOWLEDGEMENTS
`This work was supported by grant GM28013 from the National
`Institutes of Health and by grant NP-704 from the American
`Cancer Society.
`
`REFERENCES
`1. Joyce, C.M., Kelley, W.S. and Grindley N.D.F. (1982) J. Biol. Chem. 257,
`1958-1964.
`2. Lopez, P., Martinez, S., Diaz, A., Espinosa, M. and Lacks, S.A. (1989)
`J. Bio. Chem. 264, 4255-4263.
`3. Lawyer, F.C., Stoffel, S., Saiki, R.K., Myambo, K., Dnmmond, R. and
`Gelfand, D.H. (1989) J. BRil. Chem. 264, 6427-6437.
`4. Leavitt, M.C. and Ito, J. (1989) Proc. NatL. Acad. Sci. U.S.A. 86,
`4465-4469.
`5. Dunn, J.J. and Stuiier, F.W. (1983) J. Mol. Biol. 166, 477-535.
`6. RAdn, B. and Rutberg, L. (1984) J. Virol. 52, 9-15.
`7. Foury, F. (1989) J. Biol. Ceem 264, 20552-20560.
`8. Iwasaki, H., Ishino, Y., Toh, H., Nakata, A. and Shinagawa, H. (1991)
`Mol. Gen. Genet. 226, 24-33.
`9. Jung, G., Leavitt, M.C., Hsieh, J.-C. and Ito, J. (1987) Proc. Natt. Acad.
`Sd. U.S.A. 84, 8287-8291.
`10. Savilahti, H. and Bamford D.H. (1987) Gene 57, 121-130.
`11. Yoshikawa, H. and Ito, J. (1982) Gene 17, 323-335.
`12. Matsumoto, K., Takano, H., Kim, C.I. and Hirokawa, H. (1989) Gene 84,
`247-255.
`13. Spicer, E.K., Rush, J., Fung, C., Reha-Krantz, L.J., Karam, J.D. and
`Konigsberg, W.H. (1988) J. Biol Chem. 263, 7478-7486.
`14. Wong, S.W., Wahl, A.F., Yuan, P.-M., Arai, N., Pearson, B.E., Arai,
`K.-i., Korn, D., Hunkapiller, M.W. and Wang, T.S.-F. (1988) EMBO J.
`7, 37-47.
`15. Pizzagalli, A., Valsasnini, P., Plevani, P. and Lucchini, G. (1988) Proc.
`Natl. Acad. Sd. U.S.A. 85, 3772-3776.
`16. Morison, A., Arad, H., Clark, A.B., Hamatake, R.K. and Sugino, A. (1990)
`Cell 62, 1143-1151.
`
`(1987) J.
`
`Bacteriol.
`
`169,
`
`17. Boulet, A., Simon, M., Faye, G., Bauer, G.A. and Burgers, P.M.J. (1989)
`EMBO J. 8, 1849-1854.
`18. Morrison, A., Christensen, R.B., Alley, J., Beck, A.K., Bernstine, E.G.,
`Lemontt, J.F. and Lawrence, C.W. (1989) J. Bacteriol. 171, 5659-5667.
`19. Gibbs, J.S., Chiou, H.C., Hall, J.D., Mount, D.W., Retondo, M.J., Weller,
`S.K. and Coen, D.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7969-7973.
`20. Kouzarides, T., Bankier, A.T., Satchwell, S.C., Weston, K., Tomlinson,
`P. and Barrell, B.G. (1987) J. Virol. 61, 125-133.
`21. Baer, R., Bankier, A.T., Biggin, M.D., Deininger, P.L., Farrell, P.J.,
`Gibson, T.J., Hatfull, G., Hudson, G.S., Satchwell, S.C., S6guin, C.,
`Tuffnell, P.S. and Barrell, B.G. (1984) Nature 310, 207-211.
`22. Davison, A.J. and Scott, J.E. (1986) J. Gen. Virol. 67, 1759-1816.
`23. Binns, M.M., Stenzler, L., Tomley, F.M., Campbell, J. and Boursnell,
`M.E.G. (1987) Nucl. Acids Res. 15, 6563-6573.
`24. Earl, P.L., Jones, E.V. and Moss, B. (1986) Proc. Natl. Acad. Sci. U.S.A.
`83, 3659-3663.
`25. Tomalski, M.D., Wu, J. and Miller, L.K. (1988) Virology 167, 591-600.
`26. Gingeras, T.R., Sciaky, D., Gelinas, R.E., Bing-Dong, J., Yen, C.E., Kelly,
`M.M., Bullock, P.A., Parsons, B.L., O'Neill, K.E. and Roberts, R.J. (1982)
`J. Biol. Chem. 257, 13475-13491.
`27. Engler, J.A., Hoppe, M.S. and van Bree, M.P. (1983) Gene 21, 145-159.
`28. Shu, L., Hong, J.S., Wei, Y.-f. and Engler, J.A. (1986) Gene 46, 187-195.
`29. Paillard, M., Sederoff, R.R. and Levings, C.S. miI (1985) EMBO J. 4,
`1125-1128.
`30. Stark, M.J.R., Mileham, A.J., Romanos, M.A. and Boyd, A. (1984) Nucl.
`Acids Res. 12, 6011-6030.
`31. Tommasino, M., Ricci, S. and Galeotti, C.L. (1988) Nucleic Acids Res.
`16, 5863-5878.
`32. Oeser, B. and Tudzynski, P. (1989) Mot. Gen. Genet. 217, 132-140.
`33. Kempken, F., Meinhardt, F. and Esser, K. (1989) Mol. Gen. Genet. 218,
`523-530.
`34. Tomasiewicz, H.G. and McHenry, C.S.
`5735-5744.
`35. Lancy, E.D., Lifsics, M.R., Munson, P. and Maurer, R. (1989) J. Bacteriol.
`171, 5581-5586.
`36. Hammond, R.A., Barnes, M.H., Mack, S.L., Mitchener, J.A. and Brown,
`N.C. (1991) Gene 98, 29-36.
`37. Mashge, A., Nishikawa, K., Ooi, T., Seto, Y. and Yamaguchi, M. (1987)
`J. Biol. Chem. 262, 8960-8962.
`38. Abbotts, J., SenGupta, D.N., Zmudzka, B., Widen, S.G., Notario, V. and
`Wilson, S.H. (1988) Biochemistry 27, 901-909.
`39. SenGupta, D.N., Zmudzka, B.Z., Kumar, P., Cobianchi, F., Skowronski,
`J. and Wilson, S.H. (1986) Biochem. Biophys. Res. Com 136, 341-347.
`40. Peterson, R.C., Cheung, L.C., Mataliano, R.J., White, S.T., Chang, L.M.S.
`and Bollum F.J. (1985) J. Biol. Chen. 260, 10495-19502.
`41. Koiwai, O., Yokota, T., Kageyama, T., Hirose, T., Yoshida, S. and And,
`K.-i. (1986) Nulc. Acids Res. 14, 5777-5792.
`42. Argos, P., Tucker, A.D. and Phillipson, L. (1986) Virology 149, 208-216.
`43. Larder, B.A., Kemp, S.D. and Darby, G. (1987) EMBO J. 6, 160-175.
`44. Hall, J.D. (1988) Trends Genet. 4, 42-46.
`45. Reha-Krantz, L.J. (1988) J. Mol. Biol. 202, 711-724.
`46. Bemad, A., Zaballos, A., Salas, M. and Blanco, L. (1987) EMBO J. 6,
`4219-4225.
`47. Wang, T.S.-F., Wong, S.W. and Kom, D. (1989) FASEB 3, 14-21.
`48. Bernad, A., Blanco, L., Lazaro, J.M., Martin, G. and Salas, M. (1989)
`Cell 59, 219-228.
`49. Polesky, A.H., Steitz, T.A., Grindley, N.D.F. and Joyce, C.M. (1990) J.
`Biol. Chem. 24, 14579-14591.
`50. Ito, J. and Braithwaite, D.K. (1990) Nucl. Acids Res. 18, 6716.
`51. Blanco, L., Bernad, A. and Salas, M. (1991) Nucl. Acids Res. 19, 955.
`52. Kornberg, A. (1974) DNA replication. W.H. Freeman and Co., San
`Francisco.
`53. Bonner, C.A., Hays, S., McEntee, K. and Goodman M.F. (1990) Proc.
`Natl. Acad. Sci. U.S.A. 87, 7663-7667.
`54. Chen, H., Lawrence, C.B., Bryan, S.K. and Moses, R.E. (1990) Nucl. Acids
`Res. 18, 7185-7186.
`55. McHenry, C.S.(1985) Mol. Cell. Biochem 66, 71-85.
`56. Shepard, D., Oberfelder, R.W., Welch, M.W. and McHenry, C.S. (1984)
`J. Bacteriol. 158, 455-459.
`57. Scheuerman, R., Tam, S., Burgers, P.M.J., Lu, C. and Echols, H. (1983)
`Proc. Natl. Acad. Sci. U.S.A. 80, 7085-7089.
`58. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci.
`U.S.A. 74, 5463-5467.
`59. Dorsky, D.I. and Crumpacker, C.S. (1990) J. Virol. 64, 1394-1397.
`60. Bemad, A., Lizaro, J.M., Sles, M. and Blano, L. (1990) Proc. Natl. Acad.
`Sci. U.S.A. 87, 4610-4614.
`
`

`

`61. Jung, G., Leavitt, M.C., Schultz, M. and Ito, J. (1990) Biochem. Biophys.
`Res. Comm. 170, 1294-1300.
`62. Neville, M.N. and Brown, N.C. (1972) Nature New Biol. 240, 80-82.
`63. Tsurumi, T., Maeno, K. and Nishiyama, Y. (1987) Gene 52, 129-137.
`64. Dekker, B.M.M. and Van Ormondt, H. (1984) Gene 27, 115-120.
`65. Beck, P.J., Gonzalez, S., Ward, C.L. and Molineux, I.J. (1989) J. Mol.
`Biol. 210, 687-701.
`66. Paces, V., Vlcek, C., Urbanek, P. and Hostomsky, Z. (1985) Gene 38,
`45-56.
`67. Smith, H.O., Annau, T.M. and Chandrasegaran, S. (1990) Proc. Natl. Acad.
`Sci. U.S.A. 87, 826-830.
`68. Doolittle, R.F.and Feng, D.-F. (1990) In Doolittle, R.F. (ed.), Methods in
`Enzymol. -Molecular Evolution: Computer Analysis of Protein and Nucleic
`Acid Sequences. Academic Press, New York, Vol. 183, pp. 659-669.
`69. Devereux, J., Haeberli, P. and Smithies, 0. (1984) Nucl. Acids Res. 12,
`387-395.
`
`Nucleic Acids Research, Vol. 19, No. 15 4047
`
`Classification of DNA polymerases
`
`A.
`
`Family A DNA po
`
`e
`
`1.
`
`Bacteral DNA p ase
`
`References
`
`a)
`b)
`c)
`
`c ca DNA poiynmrasc I
`S&Wwcccus p_m cniae DNA polymerase I
`aquadcu DNA polymerase I
`Themr
`
`2.
`
`Bacteriophage DNA pomerases
`
`a)
`b)
`c)
`
`TS DNA polymerase
`17 DNA polymerase
`Spo2 DNA polymerase
`
`3.
`
`Mltocho l DNA p merase
`
`Yeast mitochondrial DNA polymerase (MIPI)
`
`B.
`
`Family B DNA pom
`
`1.
`
`2.
`
`3.
`
`4.
`
`S.
`
`Bacterial DNA pobm
`
`E- ci DNA polymerase II
`Bacteriophage DNA polymera
`
`PRDI DNA polymerase5
`merase
`#29 DNA p
`M2 DNA polymerase
`rase
`T4 DNA p
`
`a)
`b)
`c)
`d)
`Ekarysodc DNA polymeaes
`Human DNA polymerase alpha
`a)
`Yeast DNA polymerase I
`b)
`Yeast DNA polmeras
`II
`c)
`Yeast DNA poymerase HI
`(delta)
`d)
`Yeast DNA polymerase Rev3
`e)
`Viral DNA polymases
`
`a)
`b)
`c)
`d)
`e)
`f)
`g)
`
`Herpes-1 DNA polymerase
`Human cytomeplovirus DNA polymerase
`Epstein-Barr virus DNA polymerase
`Variceila-Zoster virus DNA polymerase
`Fowlpox virus DNA polymerase
`Vaccinia virus DNA polymerase
`Autographa californica nuclear
`polyedrosis virus (AcMNPV) DNA polymerase
`Adenovrus-2 DNA polymerase
`Adenovirus-7 DNA polymerasel
`Adenovirus-12 DNA polymerase*
`Eukaryotic linear DNA plasmid encoded DNA polymerases
`
`h)
`i)
`j)
`
`a)
`b)
`c)
`d)
`e)
`
`S-1 maize mitochondrial DNA polymerase
`Kkqyvw
`es lacdis plasmid pGKLI DNA polymerase
`Kyveryces lacts plasmid pGKI2 DNA polymerase
`Claicep pwpura plasmid pCLKU DNA polymerase
`Ascobolu unmeu plasmid pAI2 DNA polymerase
`
`C
`
`Family C DNA polymerases
`
`Bacterial replicative DNA polymerases
`E coi DNA polymerase III a subunit
`yphmwiwu DNA polymerase III a subunit
`Sabnonel
`Bacius subi DNA polymerase I
`
`a)
`b)
`c)
`
`(1)
`(2)
`(3)
`
`(4)
`(5)
`(6)
`
`(7)
`
`(8)
`
`(9,10)
`(11)
`(12)
`(13)
`
`(14)
`(15)
`(16)
`(17)
`(18)
`
`(19)
`(20)
`(21)
`(22)
`(23)
`(24)
`
`(25)
`(26)
`(27)
`(28)
`
`(29)
`(30)
`(31)
`(32)
`(33)
`
`(34)
`(35)
`(36)
`
`D.
`
`Family X DNA p a
`
`a)
`b)
`c)
`d)
`e)
`
`Rat DNA polymerase ^
`Human DNA polymerase 8
`(TdT)
`Human terminal deaxynucleotidyltransferase
`Bovine terminal deaoynucleotidyltransferase (TdT)
`Mouse terminal deoxynud1eotiltransferase (dT)
`
`(37)
`(38,39)
`(40)
`(41)
`(41)
`
`Table 1. The main families and subclassifications of DNA polymerases. Those
`DNA polymerases marked with a star (*) are protein-primed DNA polymerases.
`
`

`

`4048 Nucleic Acids Research, Vol. 19, No. 15
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