Interacting Fidelity Defects in the Replicative DNA Polymerase of Bacteriophage RB69

Abstract

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase α class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo− enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr567(PolY567(A/S/T) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr567 is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the PolY567A Exo− enzyme generates mispairs more frequently but extends them less efficiently than does a Pol+ Exo− enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43. The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase α class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo− enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr567(PolY567(A/S/T) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr567 is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the PolY567A Exo− enzyme generates mispairs more frequently but extends them less efficiently than does a Pol+ Exo− enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43. base pair substitution polymerase chain reaction Bacteriophage RB69 is a relative of phage T4, with which it shares many similarities in genetic organization (1Russell R.L. Speciation among the T-Even Bacteriophages. University Microfilms Inc., Ann Arbor, MI1967Google Scholar, 2Russell R.L. Huskey R.J. Genetics. 1974; 78: 989-1014Crossref PubMed Google Scholar) and structures and functions of the phage-encoded DNA replication proteins (3Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 4Yeh L.-S. Hsu T. Karam J.D. J. Bacteriol. 1998; 180: 2005-2013Crossref PubMed Google Scholar). Replication fidelity in T4 and presumably also in RB69 is determined almost exclusively by the fidelities of the phage-encoded DNA polymerase and its associated proofreading 3′-5′ exonuclease (5Drake J.W. Ripley L.S. Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: 98-124Google Scholar). This useful simplicity reflects the fact that T4 DNA replication appears to be devoid of DNA phage T4 is to the of the Escherichia J.W. Genetics. PubMed Google Scholar) and to mutational on its for T4 mutator mutations to for the of in and the mutational to base does the in (5Drake J.W. Ripley L.S. Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: 98-124Google Scholar). The DNA polymerases of T4 and RB69 of phage gene are of the polymerase α or B family of DNA which the replicative polymerases and of and the polymerases of of their DNA J. PubMed Scopus Google Scholar). also B family enzymes PubMed Scopus Google Scholar, J. Full Text Full Text PDF Scopus Google Scholar, L.S. J. Biol. PubMed Scopus Google Scholar). T4 gp43 and RB69 gp43 are for of of replication by this of of the of the phage to genetic and C.-C. Karam J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, in Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: Scholar, Genetics. 1998; PubMed Google Scholar, Genetics. 1998; PubMed Google Scholar). A determined of RB69 gp43 and J. Karam J.D. Full Text Full Text PDF PubMed Scopus Google Scholar). This is in the and a for the of DNA polymerase with the DNA with and with proteins of the DNA Biol. 1998; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). are for the and of replicative DNA RB69 gp43 and T4 gp43 at of their but are similar in and function can for to phage DNA replication in (3Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In RB69 gp43 has almost the and fidelity T4 gp43 the T4 in (3Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). can of of RB69 to the of its in T4 DNA replication. T4 gp43 can the fidelity of DNA replication in Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: the the T4 and RB69 gp43s a on the RB69 enzyme a is for RB69 gp43 but for T4 gp43. is to be gp43 of the to be in this conserved in that are to be by of the complementation is that a phage and a plasmid the T4 gene is the plasmid the RB69 gene C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). In is more to be the RB69 enzyme than the T4 enzyme Biol. 1998; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). and many DNA polymerases control fidelity a Pol and a 3′-5′ proofreading exonuclease Exo replicative DNA polymerases Pol and Exo the for the gp43 in of the J. Karam J.D. Full Text Full Text PDF PubMed Scopus Google Scholar). is that the of the Exo function is to by the Pol but it is less the Pol function in the and the enzyme the Exo function to been of T4 gp43 and of the polymerase family DNA Pol and Exo in the PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). assays with enzymes that the fidelity of the Pol function is that proofreading and that at or the Pol PubMed Scopus Google Scholar). The is selection of at the and the is a base at the The base selection a to fidelity and appears to on base pair and on the of the PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The strongly on the of the PubMed Scopus Google Scholar) and may the for the to the Exo for proofreading PubMed Scopus Google Scholar). and that the to proofreading a in the enzyme a to This the to its relative to the Exo Full Text Full Text PDF PubMed Scopus Google Scholar, T. 1998; PubMed Scopus Google Scholar). a of at the of the and of RB69 gp43 in Karam J. PubMed Scopus Google Scholar). of to a in with the base of the the of the for We show that at this residue can replication effects on DNA and phage RB69 gp43 with the substitution is in in the mutator of is by a RB69 that is also in the Exo function the of a does phage it does of the of DNA with the proofreading the mutation the by the appears to be to on of the in mutational by Exo− gp43 and at the gp43 Tyr567 with in of the that Tyr567 is determinant of base selection by RB69 gp43. The similarities gp43 and DNA polymerases of the B family PubMed Scopus Google Scholar, J. Full Text Full Text PDF Scopus Google Scholar, L.S. J. Biol. PubMed Scopus Google Scholar) may reveal the of at the of T4 and the T4 gene to with at and were C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). mutations in J.D. J. PubMed Google Scholar). T4 rII J.W. J. Biol. Scopus Google Scholar) were used for reversion a mutation that by the of base pair a of base are a base pair substitution that can by also at a mutation that can to by and the reversion rII mutation with been C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). efficiently T4 it the with T4 rI and also with a of T4 but with T4 to the of T4 rII Scopus Google used for DNA a plasmid RB69 gene 43 a of this in a in which is is it RB69 gp43 to than is the replication is in T4 with a of gp43 J.D. J. PubMed Google Scholar, PubMed Scopus Google Scholar). the phage and gene 43 is (3Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). with and and been J.W. Genetics. PubMed Google Scholar, J.W. Genetics. 1998; PubMed Google Scholar). T4 were for were by the were The of RB69 gp43 and Karam J. PubMed Scopus Google Scholar). for genetic were in in a at were at or rII were on the of were used to assay rII were used to assay with or rII were used to for the rI a RB69 Pol+ the control of the of C.-C. Karam J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar). derivatives of were by and by and are by of the Pol Exo PolY567A gp43 PolY567A plasmid gp43 plasmid gp43 plasmid Pol+ gp43 and plasmid PolY567A Exo− gp43 were by C.-C. and were by the to be in of a plasmid were at with to in at of to of of T4 phage of and at at a specific of in and were T4 DNA is the of to at in were with of or T4 in of the were in and at and were at by the of to the at DNA in in the were at with to in at of were to of of T4 phage of and at at a specific of were at to determine of T4 DNA the were and with T4 phage to of at at by the in The were by in the and in of and their DNA by the Inc., of DNA were for at with of a The were to for at in a in a The with and and the DNA were with a of that were to and for in this in the rII of T4 rII were in the were by on and the phage were by on are the for for is of the J.W. Genetics. PubMed Google Scholar). in T4 in with among by on The rI gene is a for a mutation gene J. J. Genetics. 1998; PubMed Google Scholar, J.W. J. Bacteriol. PubMed Google Scholar). to be in DNA it is of base the for and it a for many mutations in this for rI of T4 in their of the The were in of a of and were on Pol+ to and the were rI for of the by the of rI among determined by The the of rI among among the gene 43 phage PolY567A Exo− the mutator has a mutation that are mutationally in which the and polymerases were to in which the rI been replicated by the mutator gp43. The gp43 by a plasmid RB69 gp43 a RB69 with T4 T4 gp43 the cognate phage gene. a plasmid Pol+ or PolY567A Exo− were to and were by to T4 43 to of phage and were at on a the in the of of phage a with and to the of the in with and the for and for on Pol+ The of to In the of mutational T4-infected to the is the mutation of the rI gene is the mutation is the is the the J.W. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The mutation base is the of mutations to mutations of the of mutation We that mutations are but that mutations are efficiently among mutations and many mutations J.W. PubMed Scopus Google Scholar). B the of mutations that are and the for the of The Pol+ rI of which the T4 is of a mutation J.W. PubMed Scopus Google Scholar). this is than those in of a of J.W. PubMed Scopus Google that the many were in of and the rI by and The and the for and The of of at at at with a of at polymerase were with the with the mutation by in for and were determined Karam J. PubMed Scopus Google Scholar). were by with the and used of were for the base in is the base to pair with the or for for and and for the and its cognate base are in and The at of RB69 gp43 in T4 is conserved in of B family DNA polymerases J. PubMed Scopus Google Scholar, C.-C. Karam J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that this residue with or replication functions used to a plasmid or of RB69 gene and DNA and phage were A and The Exo− on DNA and phage The substitution the of DNA in phage The and DNA and phage less In to alanine, serine, and at at conserved to Tyr567 in the DNA and/or phage and and Karam J. PubMed Scopus Google Scholar). the substitution to DNA replication. In gp43 Exo− in assay The of by its and by the gene 43 at the Tyr567 is among conserved RB69 gp43 to in a The PolY567A Exo− DNA by a but phage the gp43 in RB69 also the of T4. This has can be by RB69 gp43 that are in polymerase but the to the of the gene 43 by the T4 (3Wang C.-C. Yeh L.-S. Karam J.D. J. Biol. Chem. 1995; 270: 26558-26564Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, C.-C. Karam J.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This PolY567A Exo− enzyme can show the PolY567A Exo− enzyme has fidelity that of the it used to the of DNA in were among the used in this or In the PolY567A Exo− enzyme appears to more DNA of than does the Pol+ enzyme. We that DNA is in phage with similar in reversion of specific mutations can reveal in mutation mutational this that by in a of base or by base pair at a base pair or The results are in Tyr567 by serine, or alanine, the mutator for mutations in and mutator for at a and The polymerases with or threonine to be more to mutator than the polymerase with In to Pol a in exonuclease function strongly mutational that the in are and for are of than for but relative are similar to relative of mutator of RB69 DNA are and in is the relative to the The of used to determine the were and for Pol+ and for in a are and in is the relative to the The of used to determine the were and for Pol+ and for We used forward mutation in the gene to determine the mutational by the RB69 gp43 mutator In to reversion which to forward mutation by at specific mutation can also reveal of mutations that are in reversion the polymerases that of T4 DNA and phage it to and to of for gp43 PolY567A which to the of a in which T4 by T4 gp43 Pol+ and RB69 gp43 The results of by this of gp43s in The of the among the by the of DNA by the gp43s is the DNA in the of the been by the gp43. We that rI are in that were by Pol+ the rI their by T4 has a of base but less is that the were by of by the enzyme. a of are to be of of a RB69 DNA polymerase on and PolY567A in a the of mutations of in Pol+ Pol+ PolY567A and mutations and/or (5Drake J.W. Ripley L.S. Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: 98-124Google Scholar) were are by mutator of rI mutations by RB69 of in a The Pol+ mutational a of a of of mutations in in the in that mutation or of The many the the and are in the Pol+ in the Pol+ Exo− gp43 proofreading appears to more efficiently to mispairs than mispairs and or more efficiently to than that T4 mutations are to results are in with the reversion The PolY567A almost exclusively of with a in of This is with the reversion The PolY567A Exo− appears to be in the and PolY567A or mutations are examined the PolY567A and Exo− mutator the of by PolY567A to in the Exo− and or mutations been appears that mutator of at Tyr567 in the were in the forward mutation their mutational and their were were In reversion more mutations with and than with PolY567A This is in the forward mutation for and also to a of to than PolY567A for for and can the of mutations but to determine the of of of RB69 DNA polymerases at of polymerases are at in a polymerases are at In of the mutations among the were The of and is with the of the T4 The of that polymerases and mispairs less or mispairs less efficiently than and mutations in of base reveal at are but this is a of than at the of almost are of may also with the of by of are the of or DNA replication. of or more base in phage T4 J. 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In the is a the and the more than of this is at but does mutations to of a at the of this a which The is in the is the and and the is a less and The with base the of base in the rI the of and a of is the of has been by mutation The T4 has and and is of and this also appears in the in T4 of the of rI a and We examined for T4 by T4 J.W. J. Bacteriol. PubMed Google Scholar, Ripley L.S. Genetics. 1998; PubMed Google Scholar) and the and to its and frequently and this for almost of in T4 in The of may in a for the of base with base the of to the Pol to the Exo J. Biol. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus (59) Google Scholar). the of and to is in the than in the Exo− at and at and for the and and the of to reveal a in T4 is to of the of in vitro a function of determined mutation can be a mutation mutations that the of replication is DNA and the of the been the The to the are for the gp43 for gp43 PolY567A the which to a mutational also to the PolY567A Exo− mutation of the of the mutations are mutations many base in and the of to be is the the is the of with rI mutations and is the can be the of and mutations among and can be with of to obtain the mutation this is the of The of and mutations for the RB69 gp43 are in rI mutation were in The of the the rI mutation and used to obtain the Pol+ Exo− and The of used to obtain of for were to the of that the of phage the for in but that of the in the polymerase This is by the of the polymerase in of of are by of the of in the the is the The of but can We used the that are for at is that of the mutations and of the mutations among the and in the of is that of the may by can be by the of the to the for the Pol+ Exo− and PolY567A them obtain and the for PolY567A Exo− by this to obtain the this may of a its it to be of rI mutations by RB69 mutations of The were The were the in the The were to obtain the which to the to obtain the in a mutations of The were The were the in the The were to obtain the which to the to obtain the The forward mutation in in the reversion of the polymerases mutator The are to be to or less the to or of the J.W. Genetics. PubMed Google that are the forward mutation many base at of which a the relative for the Pol are than in the reversion are to relative the to the of in The mutator of the Pol+ Exo− polymerase is the assay that T4 C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar) and is similar to with T4 Pol+ Exo− R.L. PubMed Scopus Google Scholar) and PubMed Scopus Google Scholar) for and for C.-C. Karam J.D. J.W. PubMed Scopus Google mutation by RB69 of is the mutation for the rI is that relative to the Pol+ and is the replication. in a is the mutation for the rI is that relative to the Pol+ and is the replication. Exo− for Pol+ and PolY567A for the and of base pair and of the a by a and a mutator or for gp43 PolY567A gp43 The for are in results are the are The with PolY567A Exo− for the base pair is of the with Pol+ This is than the relative of DNA in for the in vitro of on the of the In gp43 Pol+ Exo− strongly mispairs but more strongly mispairs than mispairs with the of than in the PolY567A is for a than for of and are in gp43 Pol+ Exo− and in gp43 PolY567A Exo− at in the of a with the of in the mutator is more strongly by a in than in and in vitro that PolY567A is a mutator with a for for by and Exo− base to be to gp43 are for in for in or for in for in mutator for for base to be to gp43 are for in for in or for in for in mutator for for in a for by and Exo− gp43 are for in for in or for in for in for for gp43 are for in for in or for in for in for for in a The for are in The for a base pair are similar for a and a were in the of the PolY567A enzyme the and the mispairs less efficiently than the Pol+ with the the A similar for the of the of DNA polymerase PubMed Scopus Google than a mutator of in this of the by the We in that a of its the Pol PubMed Scopus Google Scholar) or it for a in the inactivated Exo This the in of of the fidelity of DNA in a B family DNA determinant is the proofreading exonuclease which a of Exo determinant is the Pol in which more a residue that to be for base The of the results is than in many phage T4 is to the of the J.W. Genetics. PubMed Google Scholar) and to be to mutational on its (5Drake J.W. Ripley L.S. Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D.C.1994: 98-124Google Scholar). mutations in and Pol+ Exo− the of mutations by the Pol function in the and the with which of mutation is T4 Exo− mutations polymerase R.L. PubMed Scopus Google were to a of the on polymerase in or in vitro Karam J. PubMed Scopus Google Scholar). The Exo− a mutator the rI and and the C.-C. Karam J.D. J.W. PubMed Scopus Google Scholar) In reversion the Exo− mutation by to and by similar The DNA mutation is J.W. Genetics. 1998; PubMed Google Scholar). phage whose is to that of T4 J.W. Genetics. 1998; PubMed Google this to base pair of proofreading a fidelity of to this the fidelity of DNA be the to and by pair of fidelity for base substitution J. Biol. Chem. Full Text PDF PubMed Google Scholar) to of mutations a fidelity of a proofreading of and a of RB69 its mutation with almost the of DNA by but the in a proofreading that is than coli. DNA to the that that may the of In proofreading more strongly than more than J. Biol. Chem. Full Text PDF PubMed Google Scholar). are more and are less efficiently than by polymerases J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). T4, almost to a proofreading and The in Pol+ to in Pol+ the The for in are for mutations and for The of mutations and and the Pol+ Exo− may that at and are efficiently or that are by of proofreading in the The for by the of a base by of the to a We to the in of RB69 gp43 residue Tyr567 this residue is to the Pol L.S. J. Biol. PubMed Scopus Google Scholar, J. Karam J.D. Full Text Full Text PDF PubMed Scopus Google Scholar) and for the of were almost in a PolY567A Exo− Karam J. PubMed Scopus Google Scholar). that Tyr567 with the it does with the base than with the or it at this residue a polymerase with fidelity or a polymerase whose fidelity be RB69 gp43 Tyr567 may be related to or of A family Pol J. Karam J.D. Full Text Full Text PDF PubMed Scopus Google Scholar) and L.S. J. Biol. PubMed Scopus Google that are for at gp43 Tyr567 more than Tyr567 is more in the fidelity of base selection than in This is with the of and which are and mispairs in vitro but also of or more PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutator of RB69 gp43 and are and are more to and than is and the similar effects of and the of the substitution The mutation of the are The for of the is T4 can with this at to a of of more than of the is of whose function is for the of is at among the many of are and selection mutations the of a the for the mutator is it to the in this it is than a that does the a of is than the mutator in PolY567A Exo− were in which the mutation be of the of replicative DNA effects to fidelity for and mutationally the them to be a mutator than a of proofreading the of mutations that were J. PubMed Scopus Google Scholar). In phage T4, the effects of Exo polymerase R.L. PubMed Scopus Google Scholar) and the of that to the Pol and Exo Genetics. 1998; PubMed Google Scholar) the Exo and Pol In the Pol and Exo been examined the of that PolY567A and Exo− a more strongly than the is of the with the mutation is a than the of the the of the does We a similar mutator the polymerases are in and J. In a that the of mispairs the Pol and Exo is determined by more than and and in it that in can results be in DNA polymerases more than PubMed Scopus Google to the Exo gp43 Tyr567 strongly in the Exo be to the PolY567A enzyme the PolY567A PolY567A Exo− mutational and be which are and of reveal a reveal a Tyr567 the DNA the and the be to with a gp43 in a mutations be the in the Exo the of of the to the Pol the be the the mutations The Exo fidelity at the Pol is of a and it be to to gp43 in vitro to in vitro of polymerase fidelity the in the replication in to This to the is also to on gp43 in the with a and The is a of to a of mutationally and mutationally We and for many the of the and for a of the