Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4465-4469, June 1989 Biochemistry T5 DNA polymerase: Structural-functional relationships to other DNA polymerases (DNA polymerase I/proofreading/processivity/evolution) MARK C. LEAVITT AND JUNETSU ITO Department of Microbiology and Immunology, University of Arizona Health Sciences Center, Tucson, AZ 85724 Communicated by Lester 0. Krampitz, April 10, 1989 (receivedfor review February 1, 1989) ABSTRACT T5 DNA polymerase, a highly processive sin- proceed through double-stranded regions in template sec- gle-polypeptide enzyme, has been analyzed for its primary ondary structures or supercoiled plasmid templates. structural features. The amino acid sequence of T5 DNA We present here the DNA sequence of the T5 DNA polymerase has a high degree of homology with that of DNA polymerase gene* and the deduced amino acid sequence ofits polymerase I from Escherichia coli and retains many of the product. Comparisons of the primary structure of this en- amino acid residues that have been implicated in the 3' -* 5' zyme with other DNA polymerases suggest differences that exonuclease and DNA polymerase activities of that enzyme. may account for the high processivity of this enzyme. We Alignment with sequences of polymerase I and T7 DNA poly- also demonstrate the conservation of residues thought to be merase was used to identify regions possibly involved in the intimately involved in 3' -* 5' exonuclease and polymerase high processivity of this enzyme. Further, amino acid sequence activities. Finally, two amino acid sequence segments, which comparisons ofT5 DNA polymerase with a large group ofDNA may be involved in the 3' -*5' exonuclease function of these polymerases previously shown to exhibit little similarity to enzymes, appear to be highly conserved among a wide polymerase I indicate certain sequence segments are shared variety of DNA polymerases. among distantly related DNA polymerases. These shared re- gions have been implicated in the 3' -5' exonuclease function MATERIALS AND METHODS of I, which suggests that the proofreading domains polymerase DNA Sequencing. T5 was obtained from R. Fujimura (Oak of all these enzymes may be evolutionarily related. Ridge National Laboratory, Oak Ridge, TN). Phage T5 DNA was isolated from lysates of wild-type phage. T5 Bal I Bacteriophage T5 produces its own DNA polymerase that is fragments 11 and 12 were cloned into M13mp9 or -mpl9 (6) essential for phage DNA replication (1). This DNA polymer- and a nested set of deletions were created using the method ase is unusual in that it is highly processive; it extensively of Dale (7). Both strands were sequenced using either Se- elongates a primer before disassociating from the primer quenase (United States Biochemical) or the method of template and is capable of strand displacement. T5 DNA Maxam and Gilbert (8). A phage DNA fragment that included polymerase is more processive than any other single- the Bal I fragment 11-12junction was sequenced to eliminate polypeptide DNA polymerase on comparable templates (2, the possibility of a small intervening Bal I fragment at this 3). Similarly, its 3' -* 5' exonuclease or proofreading activity location. will processively hydrolyze hundreds of nucleotides before Amino Acid Sequence Comparisons. Protein similarity disassociation using either double- or single-stranded DNA searches of the National Biomedical Research Foundation substrates (4). The structural characteristics that confer high protein sequence library were performed (Release 16, March processivity upon polymerases are currently not understood, 1988) using FASTA (9). Amino acid sequence comparisons or perhaps because other well-studied DNA polymerases re- alignments were accomplished using LFASTA (9) or BESTFIT quire additional proteins to become processive. In contrast, or GAP (from the University ofWisconsin Genetics Computer processivity is an intrinsic property of T5 DNA polymerase Group). and therefore it is an appropriate subject for investigation in the area of DNA replication. Furthermore, this polymerase is also rare in its ability to RESULTS utilize nicked circular duplex DNA as a template and can DNA and Amino Acid Sequences of T5 DNA Polymerase. unwind the parental DNA strand from its template as it The physical location of the T5 DNA polymerase gene has synthesizes the new DNA strand from the 3'-OH end of the been identified (10) by restriction fragment rescue of poly- nick. The only other DNA polymerase capable of using a merase amber mutants, as being within the region of Bal I nicked template or of strand displacement in the absence of restriction fragments 11 and 12 at 58.3-61.3% of the distance other protein factors is Escherichia coli DNA polymerase I from the left end of the genome. DNA sequence analysis of (Pol I) or its large (Klenow) proteolysis product (4, 5). The Bal I fragments 11 and 12 reveals an open reading frame of dual properties of high processivity and strand displacement 2487 nucleotides and suitable ribosome binding site that may make T5 DNA polymerase well suited for use in dide- would code for a protein of 94.3 kDa (Fig. 1). This is in rough oxynucleotide DNA sequencing. The high processivity of agreement with the predicted molecular mass of T5 DNA modified T7 DNA polymerase-thioredoxin complex, known polymerase (96 kDa), as estimated by SDS/polyacrylamide commercially as Sequenase, has made it very popular for use gel electrophoresis (13), and is consistent with the direction in DNA sequencing projects. Additionally, the strand-dis- of transcription of the T5 DNA polymerase gene, as deter- placement ability of T5 DNA polymerase may enable it to mined by Schneider et al. (12). The publication costs of this article were defrayed in part by page charge Abbreviation: Pol l, DNA polymerase I. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M24354). 4465 Downloaded by guest on September 26, 2021 4466 Biochemistry: Leavitt and Ito Proc. Natl. Acad. Sci. USA 86 (1989) BalI map of T5 genome (121 kb) 1 4 9 10 7 5 6 1211 8 2 13 3 I I I I I I I I I . 0.5 kb A polymerase gene rTMi BalIji BalI HpaI EcoRI BalI SmaI RBS -35 -10 -1 ATCTATICCATATCIW~ ~ ~ ~ ~ ~ ~ ~ ~ a ATA7AGATl~fTATrAG 120 M Y S I C V T R S C PPVV V CCSS K KKHH I TIT I C TPT P ENNPP F DPD PN DYD Y D FVIF V I LYL V C 40 aMYS=AI~CVCTRSC 240 A E P FL Y F A G K K G I G D Y T G K R V E Y N G YA N W I A S I S P A Q L H F 80 360 K P E M K P V F D A T V E N I H D I I N G R E K I A KA G D Y R P I'T D P D E A 120 480 E E Y I K M V Y N M V I G P V A F D S E T S A L Y C R D G Y L L C V S I S H Q E 160 600 Y Q C V Y I D S D C L T E V A V Y Y L Q K I L D S E N H T I V F H N LK F D M H 200 TATMCTACC llNCGAAIClAMOCACAT_A_A A 720 F Y K Y HL G L T F D K A H K E R R L H D T M L Q H Y L D E R R G T H G L K S 240 840 L A M K Y TD M G D Y D F E L D K F K D D Y C K A H K I K K E DF T Y D L I P F 280 . Ba1I 960 D I M W P Y A AK D T D A T I R L H N F F L P K I E K N E K L CS L Y Y D V L M 320 1080 P G C V F L Q R V E D R G V P I S I D R L K E A Q Y Q L T H N L N K A R E K L Y 360 1200 T Y P E V K Q L E Q D Q N E A F N P N S V K Q L R V L L F D Y V G L T P T G K L 400 1340 T D T G A D S T D A E A L N E L A T Q H P I A K T L L E I R K L T K L I S T Y V 440 1440 E K I L L S I D A D G C I R T G FH E H M T T S G R L S SSS K L N L Q Q L P R 480 1560 D E S I I K G C V A P P G Y R V I A W D L T T A E V Y Y A A V L S G D R N M Q 520 1680 Q V F I N M R N E P D K Y P D F H S N I A H M V F K L Q C E P R D V K K L F P A 560 1800 L R Q A AKA I T F G I L Y G S G P A KV A H S V N E A L L E Q AA K T G E P F 600 1920 V E C T V A D A K E Y I E T Y F G Q F P Q L K R W I D K C If D Q I K NH G F I Y 640 2040 S H F G R K R R L H N I H S E D R G V Q G E E I R S G F N A I I Q S A S S D S L 680 CmrA 1111 2160 L L G A V D A D NE I I S L G L E Q E M K I V M L V H D S V V A I V R E D L I D 720 CAATACMSAAMAAA~cC~r~trAATA~h~AGAAAG~c1'iarA'c1AcUI X 2280 GIS I P GC P I G I D S D S E A G G S R D Y S C 760 Q Y N E I L I R N I Q K D R EcoRI.