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A novel family of DNA-polymerase-associated B subunits

A novel family of DNA-polymerase-associated B subunits
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  TIBS 24 –  JANUARY 1999 14 0968–0004/99/$ – See front matter © 1999, Elsevier Science. All rights reserved. PII: S0968-0004(98)01327-9 A novel family of DNA-polymerase-associatedB subunits DNA-dependent DNA polymerases fallinto class A, B or C, on the basis of theirsequence similarity with the products ofthe  Escherichia coli  genes polA, polB and polC, respectively (which encodeDNA polymerases I, II and III,respectively), or into class X, which hasno representative in  E. coli  1 . Class B is thelargest of these classes, including mosteukaryotic and archaebacterial DNA polymerases as well as many viraland bacteriophage enzymes. Four of thesix DNA polymerases characterized in  Saccharomyces cerevisiae fall into class B:the DNA polymerases  ,  and  , which are required for replication 2 ; andDNA polymerase  , which is required forDNA synthesis past sites of templatedamage during replication 3 . The catalyticsubunits of these eukaryotic DNA polymerases are generally associatedwith at least one smaller polypeptide: the B subunit. Genetic evidence indicatesthat, in  S. cerevisiae and  Schizosaccharomyces pombe , the Bsubunits of DNA polymerases  ,  and  are essential for viability 2,4–6 . Noenzymatic activities have been assignedto these polypeptides, and they do notcontain any known sequence motifs. B subunits have been implicated instabilization of the catalytic subunit 5,7 ,mediation of protein–proteininteractions 6,8 , and cell-cycle control andregulation 9,10 .We used the sequence of the recentlycloned B subunit of the human DNA polymerase  11,12 to search for related PROTEIN SEQUENCE MOTIFS  TIBS 24 –  JANUARY 1999 15 proteins in the non-redundant database atthe NCBI, using PSI-Blast 13 . Within fiveiterations, we retrieved the B subunits ofthe eukaryotic DNA polymerases  ,  and  , and three predicted open readingframes from Archaea (   p  10  9  ). Inaddition, we found a fourth archaealhomologue, the B subunit of a recentlydiscovered  Pyrococcus furiosus DNA polymerase II, which does not seem tobelong to class B (Ref. 14). No falsepositives were apparent in thesesearches. We further established thesimilarities by performing reciprocalsearches with other B subunits and byusing PROFILESEARCH 15 , and therebydefined a new protein family. We furtheraligned the sequences by using PIMA  16 and MACAW 17 , refining them on the basisof secondary-structure predictions madeby the PHD program 18 and subfamilyalignments. The similarity within the B-subunit family is most prominent in theC-terminal portion, whereas no clearhomology can be detected in the N-terminus.The multiple-sequence-alignmentanalysis revealed the presence of 12conserved motifs (Fig. 1). Furthermore, aproline-rich region is present betweenmotifs III and IV. Individual secondary-structure predictions for thesingle family members, for the foursubfamilies and for the entire familysuggest that the B subunits all have asimilar fold. Notably, the mutationsdescribed for B subunits coincide in mostcases with conserved residues or areas ofconserved structure 4,6,10,19 (Fig. 1). Theconserved regions probably form crucialstructural and functional sites.Phylogenetic analysis of the familymembers indicates that the B subunitsfall into two subgroups: the firstrepresents DNA polymerases  and  ; thesecond represents DNA polymerase  and the archaeal proteins (Fig. 2). The B subunits might have srcinated from a single ancestor that existed before the divergence of archaea and theeukaryotes. The eukaryotic B subunitswould then have evolved by twosubsequent gene-duplication events.Alternatively, two B subunits might haveexisted already (prior to the branching ofthe two domains); one of them could thenhave been lost during the evolution ofarchaea, while duplication in theeukaryotes gave rise to the DNA polymerase  and  subunits.Interestingly, the corresponding largesubunits of the DNA polymerases inarchaea and eukaryotes are notrelated 14,20 . The only conserved motifidentified in  P. furiosus DNA polymerase IIis a putative zinc finger, which is similar PROTEIN SEQUENCE MOTIFS Figure 1 (facing page) Multiple alignment of B subunits associated with DNA polymerases (Pols). Motifs common to all proteins are indicated by Roman numerals. Aminoacids are coloured according to the degree of conservation (100%,red;  80%,green;  60%,yellow). The consensus includes residues conserved inthe majority (  60%) of the aligned sequences [uppercase,identical; a,aromatic (F,Y and W); h,hydrophobic (L,I,V and M); n,negatively charged (Eand D); o,polar (S and T)]. Conserved  -strands (E) and  -helices (H) and the respective reliability indices are shown above the consensus (predictedby the PHD program 18 ). The numbers in the sequences indicate the position of residues and the numbers of residues between aligned blocks. Thesequence for Caenorhabditis elegans  (Ce) Pol   appears to be incomplete. Mutations described for the B subunits are underlined in the alignment;the type of mutant is given below each block (1,lethal two-amino-acid insertion 10 ; 2,temperature-sensitive point mutation 4,6 ; 3,lethal deletion 10 ; 4,temperature-sensitive two-amino-acid insertion 10 ; 5,suppressor of a temperature-sensitive mutation in the catalytic subunit 16 . Accession numbersfor the sequences follow: Methanobacteriumthermoautotrophicum (Mt) Pol (predicted),AE000903; Methanococcusjannaschii  (Mj),Pol F64387; Archaeoglobus fuldigus  (Af) Pol (predicted) AE000979; Pyrococcus furiosus  (Pf) Pol II,D84670; H. sapiens  (Hs) Pol  ,P49005; C  . elegans  Pol  (pre-dicted),Z73425; Arabidopsis thaliana (At) Pol  (predicted),AC002561; Saccharomyces cerevisiae  Pol  ,D50324; Schizosaccharomyces pombe  Pol  ,Y12561; H. sapiens  Pol  ,S39621; Drosophila melanogaster  (Dm) Pol  254200; C. elegans  Pol  (predicted),S43564; S. cerevisiae  Pol  ,P38121; Homo sapiens  Pol  ,AF036899; S. cerevisiae  (Sc) Pol  M61710; C. elegans  Pol  (predicted),U52002. Several mammalian B subunits of the DNA polymerases  ,  and  were identified in addition to the human proteins. These were not included in the alignment,because of the highdegree of similarity among the mammalian sequences. PRR,proline-rich region. Ce Pol  Sc Pol  Hs Pol  Sc Pol  Sp Pol  Sc Pol  At Pol  Hs Pol  Ce Pol  Dm Pol  Af PolMj PolMt PolHs Pol  Pf Pol ll999/998/+992/950/+1000/758/+669/926/+777/922/+1000/996/+643/305/-756/251/+530/490/+899/983/+913/954/+757/702/+0.5 aa Figure 2 Phylogenetic tree for the B subunits associ-ated with DNA polymerases. We constr-ucted the aligment by distance analysis,using the alignment shown in Fig. 1 and theNeighbour-Joining 23 option with correctionfor multiple substitution in Clustal X (Ref.24). The Caenorhabditis elegans Pol  pro-tein was excluded from the phylogeneticanalysis because the sequence appearedto be incomplete. We performed 1000 boot-strap replicates to study the confidence of the branching order. To further assess thetopology of the tree,we performed parsi-mony analysis,including 1000 bootstrapreplicates,using the programs PROTPARS,SEQBOOTand CONSENSUS of the PHYLIPpackage; maximum-likelihood analysis wasperformed with the PROTML option of theMOLPHY package. Bootstrap values fornodes are indicated in the order distance/parsimony analysis,and topologies con-firmed by the maximum-likelihood methodare indicated (  ). The tree was drawn withthe DRAWTREE program of PHYLIP. Branchlengths shown are those calculated usingthe Neighbour-Joining method. The scalebar indicates the evolutionary distance asmean number of expected amino acidresidue replacements (aa) per site.  TIBS 24 –  JANUARY 1999 16 to those present in the eukaryoticreplicative DNA polymerases  ,  and  (H. Pospiech, M. Mäkiniemi and J. E.Syväoja, unpublished). The conservationof the B subunits argues for the existenceof a conserved, fundamental functionrather than distinct, highly specific roles.What could such a function be? DNA replication is one possibility, given that alleukaryotic DNA polymerases that containconserved B subunits are involved in thisprocess 21,22 . One could therefore envisagethat B subunitsare required for theregulation of DNA polymerase activityduring replication. Alternatively, the Bsubunits might guide the polymerases tothe replication fork or maintain thestructural integrity of the catalytic subunits.In conclusion, identification of the B-subunit family could finally provide uswith the knowledge required to shed lighton the fundamental function of theseenigmatic proteins. Acknowledgements This work was supported by a grantfrom the Research Council for theEnvironment and Natural Resources,Academy of Finland. References 1 Braithwaite,D. K. and Ito,J. (1993) Nucleic Acids Res. 21,787–802 2  Sugino,A. (1995) Trends Biochem. Sci. 20,319–323 3  Nelson,J. R. et al. (1996) Science  272,1646–1649 4  Hashimoto,K. et al. (1998) Nucleic Acids Res. 26,477–485 5  Araki,H. et al. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4601–4605 6  MacNeill,S. A. et al. (1996) EMBO J. 15,4613–4628 7  Zhou,J-Q. et al. (1997) Nucleic Acids Res. 25,1094–1099 8  Araki,H. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,11791–11795 9  Nasheuer,H-P. et al. (1991) J. Biol. Chem. 266,7893–7903 10  Foiani,M. et al. (1994) Mol. Cell. Biol. 14,923–933 11 Li,Y. et al. (1997) J. Biol. Chem. 272,32337–32344 12  Jokela,M. et al. (1998) Nucleic Acids Res. 26,730–734 13  Altschul,S. F. et al. (1997) Nucleic Acids Res. 25,3389–3402 14  Uemori,T. et al. (1997) Genes Cells  2,499–512 15  Gribskov,M. et al. (1989) Methods Enzymol. 183,146–159 16  Smith,R. F. andSmith,T. F. (1992) Protein Eng. 5,35–41 17  Schuler,G. D. et al. (1991) Proteins  9,180–190 18  Rost,B. and Sander,C. (1994) Proteins  19,55–72 19  Giot,L. et al. (1997) Genetics 146,1239–1251 20  Ishino,Y. et al. (1998) J. Bacteriol. 180,2232–2236 21 Wang,T. S-F. (1996)in DNA Replication in Eukaryotic Cells  (De Pamphilis,M. L.,ed.),pp. 461–493,Cold Spring HarborLaboratory Press 22  Zlotkin,T. et al. (1996) EMBO J. 15,2298–2305 23  Saitou,N. and Nei,M. (1987) Mol.Biol. Evol. 4,406–425 24  Thompson,J. D. et al. (1994) Nucleic Acids Res. 22,4673–4680 MINNA MÄKINIEMI,HELMUT POSPIECH,SEPPO KILPELÄINENAND MAARIT JOKELA Biocenter Oulu andDept of Biochemistry,University of Oulu,FIN-90570 Oulu,Finland. MAUNO VIHINEN Institute of MedicalTechnology,University of Tampere,FIN-33101 Tampere,Finland. JUHANI E. SYVÄOJA Biocenter Oulu andDept of Biochemistry,University of Oulu,FIN-90570 Oulu,Finland.Email: PROTEIN SEQUENCE MOTIFS Can you contribute to Computer Corner? Have you come across any applications(freeware or commercially available), CDs,servers or tips recently that might be of interest toother biochemists and molecular biologists? If so,why not let us know so that we can review themin Computer Corner  ? Pete Jeffs is a freelancer working in Paris,France.
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