`
`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),
`
`Columbia Ex. 2089
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`
`
`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),
`
`
`
`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.
`
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`
`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.
`
`
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`4048 Nucleic Acids Research, Vol. 19, No. 15
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