Nucleic Acids Research, Vol. 18, No. 23 7185
Aphidicolin inhibits DNA polymerase 11 of Escherichia coli, an alpha-like DNA polymerase H.Chen', C.B.Lawrence1, S.K.Bryan and R.E.Mosesl,* Department of Cell Biology and Houston, TX 77030, USA
'institute for Molecular Genetics, Baylor College of Medicine, GenBank accession no. M35371
Submitted September 11, 1990 The polB gene of E. coli encodes DNA polymerase II. We have reported the cloning (1, 2) and sequencing of the polB gene (3) (GenBank Accession number M3537 1). DNA polymerase H appears to be a non-essential function since the polB gene is missing from some widely used strains (3). Several sub-families of DNA polymerases have been identified (4). In this communication, we demonstrate that DNA polymerase II of E. coli is an a-like DNA polymerase by amino acid sequence conservation and inhibition by aphidicolin. A search of the MBCRR Protein Pattern Library indicated significant similarity (6.3 standard deviations above the mean of comparisons with a negative control set) to only a single pattern (pattern 156) generated from a set of a-like polymerases (Fig. 1). DNA polymerase II has the highest overall similarity to human DNA polymerase ca and phage T4 DNA polymerase. The entire length of DNA polymerase II is similar to a subregion of both polymerases and within this region the sequences are both approximately 22% identical. There are three sequence motifs characteristic of the a-like family (regions I, H, III) (4, 5). The H region shown in Figure 1 corresponds to region I characterized by the presence of 5-7 contiguous conserved amino acids (YGDTDS). The E region corresponds to region II. In addition to a central conserved sequence of 5-10 residues (DSLYPS), there are additional sequence identities on both sides of the conserved center. Regions F and G correspond to region HI with invariant residues at five positions. These three regions are in the same linear arrangement (H-HI-I) as other DNA polymerases in this family and the relative distances between the regions are
similar. The N-terminal region (residues 1-30 1) of DNA polymerase II has significant similarity to only a subset of the ai-like DNA polymerases (human ca, phage T4, yeast polIHI, Epstein-Barr virus, and Autographa californica nuclear polyhedrosis virus [A-
*
To whom correspondence should be addressed
CNPV]). The regions of the six polymerases having the highest mutual similarity were aligned (shaded boxes in Figure 1). Region A in other a-like polymerases has 3'-5' exonuclease activity. Human DNA polymerase a is inhibited by the drug aphidicolin. We found that aphidicolin also inhibited DNA polymerase II (Ki = 50 ptM). The peculiar aphidicolin sensitivity of DNA polymerase H offered an opportunity to assess its possible role in DNA replication. Toluene-treated E. coli cells offer a convenient means of assessing the effect of compounds on the elongation phase of DNA replication, since the permeable cells eliminate problems of transport (6). We found no significant effect of aphidicolin on the level of ATP-dependent synthesis in seven different strains of E. coli, including two strains apparently lacking the polB gene (3), MC 1061 and MC1000. We conclude that there is no detectable role of DNA polymerase II in the elongation phase of DNA replication.
ACKNOWLEDGEMENTS This work was supported by American Cancer Society grant NP-688 and Texas Advanced Technology Program #4949-041.
REFERENCES 1. Bryan,S.K., Chen,H., Sun,Y. and Moses,R.E. (1988) Biochim. Biophys. Acta 951, 249-254. 2. Chen,H., Bryan,S.K. and Moses,R.E. (1989) J. Biol. Chem. 264, 20591 -20595. 3. Chen,H., Sun,Y., Stark,T., Beattie,W. and Moses,R.E. (1990) DNA and Cell Biol., in press. 4. Delarue,M., Poch,O., Tordo,N., Moras,D. and Argos,P. (1990) Prot.
Engineering 3, 461-467. 5. Bernad,A., Lazaro,J., Salas,M. and Blanco,L. (1990) Proc. Natl. Acad. Sci. USA 87, 4610-4614.
6. Ross,S., Sharna,S. and Moses,R.E. (1980) Mol. Gen. Genet. 179, 595-605. 7. Sliith,R.F. and Smith,T.F. (1990) Proc. Natl. Acad. Sci. USA 87, 118-122.
7186 Nucleic Acids Research, Vol. 18, No. 23 ----KVEAMALK PDLVNVIKDVSFP_PL VVMAFSMK1aMQNfAKN HQNEIIAMA2iAVHH§ FALDKAAPKPP--FQ Hu.a VKKSTA,LNQPVSWCMN YEjREG2 VT------ VYFJVRP PERYLMERFITSPVW VEGDMHNTIVNARL EPHPDYR-PPLKWV4 IDIETTRHGEjd--YC Pol II T4 RDWI_MqEDI.QLEAL GMNDFKLAYISDTYG S_E1~YDRKFV- -RVA NCDIEV72DKFPDPM LKAEYE~ID-LITHYDa IDDRFYVFDLLdNSMY Hum. a
Pal II T4 Yet PIII EBV
SHFCVVSKPKDCIFP YAFKEVIEK ---KN- -VKVEVAATERThLG
FFLAKVHK1gPM?V ............ ..............'
1---NV-CKAPHWJ
IGLEACGQRivYHLG PEI=D-ASS ---LD - -FEIEZYVASRPQ_L. KI2NAWFAN ............. ....... ..-..-YR..... ......RA~T~ 1MLGRt~F GSVSKWDAKLAAKID C!EGD -EVPQEILDR VIYMPFDNERDHL!1E
581 78 154
66 0 154 240
T
D~D ~XTGB L293 ~JRPXL SKIGRLKRSNMPKLG 2RSGFGERNAT(M-: Zc--VEISAK-ELIR CM Mi~tVQIL- KTERVVIPMENIQNM YSESSQ-L.&t-yLr4$1~r LRLGR.DNSELEWREH GFKNGVFFAQAKPJJ '"I:ID)GIEAL-KSAFFWN FS~$~~#.EXLLG EGKSIDNP NiDRMDEI DRRFAEDKPA... KRTNH~Y 3~E--KGKPD-P SPIGRVKSKLIQNMY GSKE --- IYSIEIqVS ,::Z~YLDLYKKFAFTN LP2 LSV T4 RLV! 82 4 61 ~ 2tA$ Yet PIII~LQL
Hum. aX
Pal II
74 6 243 322
4 504 EBV A~~~~NPV V~~~~~~~~~C7~~~~~PIKI~~~~~~~~ 400~~4
E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.................
335 V9 DEEQP-L DSELIIPRI KG"::HIDW ACuP. a :MPlIQWII.-5VQVAS35 PalII PHSPG~MISRG YD.~VbLDKSY GFKHPHEYIGTAPPSE SCPNW YDHQI3 IKEA A T4 .Q....-$.....SIRQIISPTI 621EDDNKKGK Pi:l MCELNR M_SRTMHQSREFLLAFENIVDKIFKQK Yet. rV1LjALQTNIFNSI R,42 599 X~1YZQ~ZS~ EBV
SIRTLID-......D....FL......FS~EHCPEVT
1
38 7
SPVZ4~~WASLY_..UQ.ZQ
Yet Pi525
3EV~~~~~~~~.......... ACNPV40
E
H
+G
9103 VTEDENSTNE-LE PDPLEMGIKPEIR--NLTVlYGTASE MiK YSNFYAK ............. PS1IVT7QEILMBTK--VQ ...48 SS~jH QIR~ALI.AQG. fl~VjTACRFDP ............GAHE3..........QW 65 VGL R KE tA I~ EF tA FG L~~~~~~Aj~. FR.......... .........I.K.E.NKC...........V ....E 72 ZSS~A~ V~F2~709 712 692"`' 3EV PESEFD--AWSEHLEV 1AIL: GVG~ T ~~~~~~~F2.:.::Z
Hum, a Pal II T4 Pil Yet
KG
T4
KKKEMDAYRLCYNREIHD .ICPf-G QG--SQE a Hum. KQDN..L....Q..L.QQ.H SQRAYAPEQL.. GGNTF II PGLPTFTTGLRTLSD~~~............ Pal P T4 LDGNKVMVLPLRE~~~~~~~~... ......SG T4
PKA~~~.ALEESIR
YKFEEY
Y LVD DRFEHLIGMT ~ST 735 4KKR
IRY GIFKVKPIHEDIAKGD AKPSDEYCSPFHIGW MTYDKHVEGVIPEIA
82
oyeaes hsfgr hw teainet fsletdrgoso h n mutil alignments betweenM 62lk1N Figur 1.ICopst=fsvrlpiws oiDN oyeaeIhmnapa(PO$UA) 4(PLBT) es (referenc or........ followinglarge .D. rGnetLcs:E polymerases..... SWISS-PROT.. Auorph ..V) aio.ianula.o.hdossvrsGnPetLcs:NANAM.) subunit (DPO3$Y....A.... pollil Epsten-Bar.viru.(DPO$ s.umarzd.n.hefiue.Seece and T4 polymerases were.. algnd.aiwsewih.NAp.yerse..ad.heco.sie.f.heto.linmns Human alpha aesae5i9h9iur.Aioacdrsn ein uing the ultipl alignmentS falsiVoyeaeswrlge rEginB mehdoPmihadSitW7.Tes any regonareunerlne. seuence inon-halfor moe of hein........ eaes()ar.nictdwiha.arw.Psto Poiinspoosdt.b.naratinalpoy coordinates are the .......number ...of..amino. acdsfom th. -trmnl.mnoacdasgie.i.teiniatd.aa.as.nty
Aphidicolin inhibits DNA polymerase 11 of Escherichia coli, an alpha-like DNA polymerase H.Chen', C.B.Lawrence1, S.K.Bryan and R.E.Mosesl,* Department of Cell Biology and Houston, TX 77030, USA
'institute for Molecular Genetics, Baylor College of Medicine, GenBank accession no. M35371
Submitted September 11, 1990 The polB gene of E. coli encodes DNA polymerase II. We have reported the cloning (1, 2) and sequencing of the polB gene (3) (GenBank Accession number M3537 1). DNA polymerase H appears to be a non-essential function since the polB gene is missing from some widely used strains (3). Several sub-families of DNA polymerases have been identified (4). In this communication, we demonstrate that DNA polymerase II of E. coli is an a-like DNA polymerase by amino acid sequence conservation and inhibition by aphidicolin. A search of the MBCRR Protein Pattern Library indicated significant similarity (6.3 standard deviations above the mean of comparisons with a negative control set) to only a single pattern (pattern 156) generated from a set of a-like polymerases (Fig. 1). DNA polymerase II has the highest overall similarity to human DNA polymerase ca and phage T4 DNA polymerase. The entire length of DNA polymerase II is similar to a subregion of both polymerases and within this region the sequences are both approximately 22% identical. There are three sequence motifs characteristic of the a-like family (regions I, H, III) (4, 5). The H region shown in Figure 1 corresponds to region I characterized by the presence of 5-7 contiguous conserved amino acids (YGDTDS). The E region corresponds to region II. In addition to a central conserved sequence of 5-10 residues (DSLYPS), there are additional sequence identities on both sides of the conserved center. Regions F and G correspond to region HI with invariant residues at five positions. These three regions are in the same linear arrangement (H-HI-I) as other DNA polymerases in this family and the relative distances between the regions are
similar. The N-terminal region (residues 1-30 1) of DNA polymerase II has significant similarity to only a subset of the ai-like DNA polymerases (human ca, phage T4, yeast polIHI, Epstein-Barr virus, and Autographa californica nuclear polyhedrosis virus [A-
*
To whom correspondence should be addressed
CNPV]). The regions of the six polymerases having the highest mutual similarity were aligned (shaded boxes in Figure 1). Region A in other a-like polymerases has 3'-5' exonuclease activity. Human DNA polymerase a is inhibited by the drug aphidicolin. We found that aphidicolin also inhibited DNA polymerase II (Ki = 50 ptM). The peculiar aphidicolin sensitivity of DNA polymerase H offered an opportunity to assess its possible role in DNA replication. Toluene-treated E. coli cells offer a convenient means of assessing the effect of compounds on the elongation phase of DNA replication, since the permeable cells eliminate problems of transport (6). We found no significant effect of aphidicolin on the level of ATP-dependent synthesis in seven different strains of E. coli, including two strains apparently lacking the polB gene (3), MC 1061 and MC1000. We conclude that there is no detectable role of DNA polymerase II in the elongation phase of DNA replication.
ACKNOWLEDGEMENTS This work was supported by American Cancer Society grant NP-688 and Texas Advanced Technology Program #4949-041.
REFERENCES 1. Bryan,S.K., Chen,H., Sun,Y. and Moses,R.E. (1988) Biochim. Biophys. Acta 951, 249-254. 2. Chen,H., Bryan,S.K. and Moses,R.E. (1989) J. Biol. Chem. 264, 20591 -20595. 3. Chen,H., Sun,Y., Stark,T., Beattie,W. and Moses,R.E. (1990) DNA and Cell Biol., in press. 4. Delarue,M., Poch,O., Tordo,N., Moras,D. and Argos,P. (1990) Prot.
Engineering 3, 461-467. 5. Bernad,A., Lazaro,J., Salas,M. and Blanco,L. (1990) Proc. Natl. Acad. Sci. USA 87, 4610-4614.
6. Ross,S., Sharna,S. and Moses,R.E. (1980) Mol. Gen. Genet. 179, 595-605. 7. Sliith,R.F. and Smith,T.F. (1990) Proc. Natl. Acad. Sci. USA 87, 118-122.
7186 Nucleic Acids Research, Vol. 18, No. 23 ----KVEAMALK PDLVNVIKDVSFP_PL VVMAFSMK1aMQNfAKN HQNEIIAMA2iAVHH§ FALDKAAPKPP--FQ Hu.a VKKSTA,LNQPVSWCMN YEjREG2 VT------ VYFJVRP PERYLMERFITSPVW VEGDMHNTIVNARL EPHPDYR-PPLKWV4 IDIETTRHGEjd--YC Pol II T4 RDWI_MqEDI.QLEAL GMNDFKLAYISDTYG S_E1~YDRKFV- -RVA NCDIEV72DKFPDPM LKAEYE~ID-LITHYDa IDDRFYVFDLLdNSMY Hum. a
Pal II T4 Yet PIII EBV
SHFCVVSKPKDCIFP YAFKEVIEK ---KN- -VKVEVAATERThLG
FFLAKVHK1gPM?V ............ ..............'
1---NV-CKAPHWJ
IGLEACGQRivYHLG PEI=D-ASS ---LD - -FEIEZYVASRPQ_L. KI2NAWFAN ............. ....... ..-..-YR..... ......RA~T~ 1MLGRt~F GSVSKWDAKLAAKID C!EGD -EVPQEILDR VIYMPFDNERDHL!1E
581 78 154
66 0 154 240
T
D~D ~XTGB L293 ~JRPXL SKIGRLKRSNMPKLG 2RSGFGERNAT(M-: Zc--VEISAK-ELIR CM Mi~tVQIL- KTERVVIPMENIQNM YSESSQ-L.&t-yLr4$1~r LRLGR.DNSELEWREH GFKNGVFFAQAKPJJ '"I:ID)GIEAL-KSAFFWN FS~$~~#.EXLLG EGKSIDNP NiDRMDEI DRRFAEDKPA... KRTNH~Y 3~E--KGKPD-P SPIGRVKSKLIQNMY GSKE --- IYSIEIqVS ,::Z~YLDLYKKFAFTN LP2 LSV T4 RLV! 82 4 61 ~ 2tA$ Yet PIII~LQL
Hum. aX
Pal II
74 6 243 322
4 504 EBV A~~~~NPV V~~~~~~~~~C7~~~~~PIKI~~~~~~~~ 400~~4
E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.................
335 V9 DEEQP-L DSELIIPRI KG"::HIDW ACuP. a :MPlIQWII.-5VQVAS35 PalII PHSPG~MISRG YD.~VbLDKSY GFKHPHEYIGTAPPSE SCPNW YDHQI3 IKEA A T4 .Q....-$.....SIRQIISPTI 621EDDNKKGK Pi:l MCELNR M_SRTMHQSREFLLAFENIVDKIFKQK Yet. rV1LjALQTNIFNSI R,42 599 X~1YZQ~ZS~ EBV
SIRTLID-......D....FL......FS~EHCPEVT
1
38 7
SPVZ4~~WASLY_..UQ.ZQ
Yet Pi525
3EV~~~~~~~~.......... ACNPV40
E
H
+G
9103 VTEDENSTNE-LE PDPLEMGIKPEIR--NLTVlYGTASE MiK YSNFYAK ............. PS1IVT7QEILMBTK--VQ ...48 SS~jH QIR~ALI.AQG. fl~VjTACRFDP ............GAHE3..........QW 65 VGL R KE tA I~ EF tA FG L~~~~~~Aj~. FR.......... .........I.K.E.NKC...........V ....E 72 ZSS~A~ V~F2~709 712 692"`' 3EV PESEFD--AWSEHLEV 1AIL: GVG~ T ~~~~~~~F2.:.::Z
Hum, a Pal II T4 Pil Yet
KG
T4
KKKEMDAYRLCYNREIHD .ICPf-G QG--SQE a Hum. KQDN..L....Q..L.QQ.H SQRAYAPEQL.. GGNTF II PGLPTFTTGLRTLSD~~~............ Pal P T4 LDGNKVMVLPLRE~~~~~~~~... ......SG T4
PKA~~~.ALEESIR
YKFEEY
Y LVD DRFEHLIGMT ~ST 735 4KKR
IRY GIFKVKPIHEDIAKGD AKPSDEYCSPFHIGW MTYDKHVEGVIPEIA
82
oyeaes hsfgr hw teainet fsletdrgoso h n mutil alignments betweenM 62lk1N Figur 1.ICopst=fsvrlpiws oiDN oyeaeIhmnapa(PO$UA) 4(PLBT) es (referenc or........ followinglarge .D. rGnetLcs:E polymerases..... SWISS-PROT.. Auorph ..V) aio.ianula.o.hdossvrsGnPetLcs:NANAM.) subunit (DPO3$Y....A.... pollil Epsten-Bar.viru.(DPO$ s.umarzd.n.hefiue.Seece and T4 polymerases were.. algnd.aiwsewih.NAp.yerse..ad.heco.sie.f.heto.linmns Human alpha aesae5i9h9iur.Aioacdrsn ein uing the ultipl alignmentS falsiVoyeaeswrlge rEginB mehdoPmihadSitW7.Tes any regonareunerlne. seuence inon-halfor moe of hein........ eaes()ar.nictdwiha.arw.Psto Poiinspoosdt.b.naratinalpoy coordinates are the .......number ...of..amino. acdsfom th. -trmnl.mnoacdasgie.i.teiniatd.aa.as.nty
