The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ 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 Tyr 567 (Pol Y567(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 Tyr 567 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 Pol Y567A 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.Bacteriophage RB69 is a relative of phage T4, with which it shares many similarities in genetic organization (1, 2) and structures and functions of the phage-encoded DNA replication proteins (3,4). 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 3Ј-5Ј exonuclease (5). This useful simplicity reflects the fact that T4 DNA replication appears to be devoid of DNA mismatch repair; phage T4 is not subject to the action of the several Escherichia coli mismatch repair systems (6) and seems unable to repair mutational heteroduplexes on its own. Screens for T4 mutator mutations have failed to uncover evidence for the involvement of mismatch repair in mutagenesis, and the mutational dose response to base analogues does not display the mismatch repair-dependent lag seen in E. coli (5).The DNA polymerases of phages T4 and RB69 (gp43, product of phage gene 43) are members of the polymerase ␣ class or B family of DNA polymerases, which includes the replicative polymerases ␣, ␦, and ⑀ of eukaryotic cells and the polymerases of several of their DNA viruses (7). Some archaeons also encode gp43-like B family enzymes (8 -10). As such, T4 gp43 and RB69 gp43 are attractive subjects for studies of mechanisms of replication by this class of enzymes, particularly because of the amenability of the phage system to combined genetic and biochemical analyses (11)(12)(13)(14). A recently determined crystal structure of RB69 gp43 reveals five discrete domains termed N, Exo, Palm, Fingers, and Thumb (15). This structure is in the "open" configuration and provides a preliminary framework for understanding the dynamics of DNA polymerase interactions with the DNA primer template, with incoming dNTPs, and ...

Collections of mutants usually contain more mutants bearing multiple mutations than expected from the mutant frequency and a random distribution of mutations. This excess is seen in a variety of organisms and also after DNA synthesis in vitro. The excess is unlikely to originate in mutator mutants but rather from transient hypermutability resulting from a perturbation of one of the many transactions that maintain genetic fidelity. The multiple mutations are sometimes clustered and sometimes randomly distributed. We model some spectra as populations comprising a majority with a low mutation frequency and a minority with a high mutation frequency. In the case of mutants produced in vitro by a bacteriophage RB69 mutator DNA polymerase, mutants with two mutations are in Ϸ10-fold excess and mutants with three mutations are in even greater excess. However, phenotypically undetectable mutations seen only as hitchhikers with detectable mutations are Ϸ5-fold more frequent than mutants bearing detectable mutations, indicating that they arose in a subpopulation with a higher mutation frequency. Excess multiple mutations may contribute critically to carcinogenesis and to adaptive mutation, including the adaptations of pathogens as they move from host to host. In the case of the rapidly mutating riboviruses, the viral population appears to be composed of a majority with a mutation frequency substantially lower than the average and a minority with a huge mutational load.bacteriophage RB69 ͉ multiple mutations ͉ mutation rate ͉ ribovirus

DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2–3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood. While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3′-5′ exonuclease activity) in the fidelity of DNA replication and to highlight what is known about switching from polymerase to exonuclease active site.

Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to non-complementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k pol and K D values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k pol / K D ) for mispaired bases relative to the wild type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 × 10 −5 µM −1 s −1 for dCMP opposite templating C to 2 × 10 −3 µM −1 s −1 for dAMP opposite templating C. These k pol /K D values are 3-60 fold greater than those observed with the wild type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting E. coli, harboring a plasmid encoding the L561A variant of RB69 pol, with T4 phage bearing a mutant rII locus and the rates of reversions to rII + were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo − mutator activities. Keywords base selectivity; transient-state kinetic parameters; mutation frequency; modeling of mismatches DNA polymerase is the central component of replicases that are responsible for faithfully copying DNA. Despite the fact that all four dNTPs are potential substrates, replicative DNA polymerases are able to limit the incorporation of mismatched bases to about one in a million [for reviews see (1-8)]. Although this error frequency is acceptable for phage replication, it is still far too high to maintain genetic integrity during cell proliferation in † Supported by National Institutes of Health, Public Health Service grants GM63276 (to W.K.) and TW006626 (to A.B.). J.W.D. was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. * To whom correspondence and reprint requests should be addressed. telephone, (203) 785-4599; fax, (203) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript more complex organisms. To increase fidelity, cells have a number of mechanisms for correcting errors, including the ability of the replicative DNA polymerase itself, or an associated subunit, to excise misincorporated bases. In addition, there are specialized DNA polymerases that confront DNA damage inflicted by radiation, oxidation, alkylating agents, etc. which block the progress of replic...