Accepted Manuscript Title: DNA polymerase 3 →5 exonuclease activity; different roles of the beta hairpin structure in family-B DNA polymerases Author: Hariyanto Darmawan Melissa Harrison Linda J. Reha-Krantz PII: DOI: Reference:
S1568-7864(15)00050-6 http://dx.doi.org/doi:10.1016/j.dnarep.2015.02.014 DNAREP 2065
To appear in:
DNA Repair
Received date: Revised date: Accepted date:
4-11-2014 12-2-2015 13-2-2015
Please cite this article as: H. Darmawan, M. Harrison, L.J. Reha-Krantz, DNA polymerase 3 rightarrow5 exonuclease activity; different roles of the beta hairpin structure in family-B DNA polymerases, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.02.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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DNA polymerase 3’→5’ exonuclease ac vity; different roles of the beta hairpin structure in family-B DNA polymerases
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Hariyanto Darmawan, Melissa Harrison, Linda J. Reha-Krantz*
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Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
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*Corresponding author at: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Tel,: +1 780 492 5383; fax: +1 780 492 9234.
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E-mail address:
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Highlights
T4 DNA polymerase proofreading requires a beta hairpin structure
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Yeast DNA polymerase delta proofreading does not require the hairpin
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The yeast DNA polymerase delta hairpin is needed for fitness
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DNA polymerase evolution
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Mismatch repair regulates the checkpoint when DNA pol delta replication is hindered
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Keywords:
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DNA polymerase proofreading
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Phosphonoacetic acid sensitivity
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Hydroxyurea sensitivity
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DNA polymerase evolution
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Mismatch repair and checkpoint response 1 Page 1 of 48
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DNA polymerase mutations in cancer
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ABSTRACT
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Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure
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that resides in the exonuclease domain. Genetic, biochemical and structural studies demonstrate
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that the phage β hairpin acts as a wedge to separate the primer-end from the template strand in
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exonuclease complexes. Single amino acid substitutions in the tip of the hairpin or deletion of
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the hairpin prevent proofreading and create “mutator” DNA polymerases. There is little known,
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however, about the function of similar hairpin structures in other family B DNA polymerases.
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We present mutational analysis of the yeast (Saccharomyces cerevisiae) DNA polymerase δ
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hairpin. Deletion of the DNA polymerase δ hairpin (hp) did not significantly reduce DNA
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replication fidelity; thus, the β hairpin structure in yeast DNA polymerase δ is not essential for
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proofreading. However, replication efficiency was reduced as indicated by a slow growth
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phenotype. In contrast, the G447D amino acid substitution in the tip of the hairpin increased
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frameshift mutations and sensitivity to hydroxyurea (HU). A chimeric yeast DNA polymerase δ
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was constructed in which the T4 DNA polymerase hairpin (T4hp) replaced the yeast DNA
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polymerase δ hairpin; a strong increase in frameshift mutations was observed and the mutant
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strain was sensitive to HU and to the pyrophosphate analog, phosphonoacetic acid (PAA). But
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all phenotypes - slow growth, HU-sensitivity, PAA-sensitivity, and reduced fidelity, were
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observed only in the absence of mismatch repair (MMR), which implicates a role for MMR in
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mediating DNA polymerase δ replication problems. In comparison, another family B DNA
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polymerase, DNA polymerase , has only an atrophied hairpin with no apparent function. Thus,
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while family B DNA polymerases share conserved motifs and general structural features, the β
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hairpin has evolved to meet specific needs.
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1. Introduction
The role of the 3’ → 5’ exonuclease activity associated with many DNA polymerases
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was revealed in two seminal papers published in 1972. Brutlag and Kornberg [1] reported that
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the 3’→5’ exonuclease activity of Escherichia coli DNA pol I was specific for mismatches at the
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3’-end of the primer strand, which suggested that the 3’ → 5’ exonuclease of E. coli DNA pol I
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functioned in error avoidance. A similar “proofreading” role was proposed for the 3’ → 5’
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exonuclease activity of bacteriophage T4, and evidence was provided by biochemical studies of
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“mutator” (increased replication errors) and “antimutator” (decreased replication errors) T4 DNA
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polymerases [2]. Speyer and Drake discovered that several alleles of the T4 DNA polymerase
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strongly altered replication fidelity by increasing or decreasing the spontaneous mutation
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frequency [3-5]. While differences in the accuracy of nucleotide incorporation could explain the
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mutator and antimutator phenotypes, Muzyczka et al. [2] found that the ratio of nuclease to
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polymerase (N/P) activity was perturbed in the mutant DNA polymerases; the N/P ratio was
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reduced for mutator DNA polymerases and increased for antimutator DNA polymerases. Further
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characterization of mutator and antimutator DNA polymerases confirmed that mutator DNA
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polymerases proofread mismatched primer-ends less efficiently while antimutator DNA
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polymerases proofread mismatched and even matched primer-ends more efficiently than
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observed for the wild type T4 DNA polymerase [2,6].
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DNA polymerase proofreading, however, requires more than removal of an incorrect
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nucleotide. The primer-end with the terminal incorrect nucleotide must be physically moved 3 Page 3 of 48
from the polymerase to the exonuclease active site where the terminal nucleotide is removed,
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then the trimmed primer-end is returned to the polymerase active site [7,8]. This is polymerase-
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to-exonuclease and exonuclease-to-polymerase switching. Many details of the proofreading
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pathway catalyzed by the T4 DNA polymerase have been revealed by a large collection of
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mutants, which have amino acid substitutions throughout the DNA polymerase [7-18].
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Proofreading is clearly a dynamic process that involves participation by all five protein domains.
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The first step in the proofreading pathway is recognition of a newly misincorporated
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nucleotide at the primer-end in the polymerase active site, and then the “decision” is made to
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extend the mismatch by incorporation of the next nucleotide or to transfer the primer-end to the
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exonuclease active site [7,19,20]. Formation of exonuclease complexes requires separation of
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the primer-end from the template strand and transfer of the primer-end to the exonuclease active
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site, which is ~40 Å from the polymerase active site. T4 and RB69 DNA polymerases require a
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β hairpin structure in the exonuclease domain for these tasks; the hairpin acts as a wedge and
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inserts between the template and primer strands (14,21; Fig. 1A). T4 DNA polymerase mutants
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with single amino acid substitutions in the tip of the hairpin, e.g. G255S, can degrade single-
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stranded DNA, but have much less activity with double-stranded DNA unless there is a
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preformed strand separation of at least three terminal base pairs [14,22]; this is because the
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G255S-DNA polymerase has reduced ability to form stable, strand-separated exonuclease
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complexes [22-24]. Single amino acid substitutions for Y254 in the T4 DNA polymerase and
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deletion of the hairpin (hp) in the RB69 DNA polymerase also produce proofreading-deficient
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DNA polymerases [25-27]. The RB69 hp mutant appears incapable of forming exonuclease
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complexes with double-stranded, primer-template DNA, but still retains exonuclease activity
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with single-stranded DNA [27]. Aller et al. [28] captured a well-ordered exonuclease complex
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structure with the RB69 DNA polymerase that confirms the role of the β hairpin structure in
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strand separation and in the stabilization of the primer-end in the exonuclease active site (Fig.
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1B). Residues in the tip of the RB69 DNA polymerase hairpin, Y257 and M256, which are
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analogous to T4 DNA polymerase residues Y254 and M253, are wedged firmly between the
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primer and template strands. Note that structural studies were performed with the phage RB69
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DNA polymerase, which is closely related in sequence (61%) and function to the phage T4 DNA
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polymerase [29]. Thus, the inability to form exonuclease complexes with primer-template DNA
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severely reduces exonuclease activity even though exonuclease activity with single-stranded
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DNA substrates is retained.
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If the mismatched primer-end cannot be extended, but exonuclease complexes also
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cannot be formed readily, then continued replication is stalled at the crossroads of the nucleotide
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incorporation and proofreading pathways and the reverse reaction, pyrophosphorolysis [20,24].
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Crossroad complexes are pre-translocation (pre-T) complexes, which are formed after nucleotide
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incorporation and dissociation of pyrophosphate, just before the DNA polymerase translocates to
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be in position to bind the next incoming nucleotide and form post-translocation (post-T)
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complexes.
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of post-T complexes is hindered if the primer-end is not correct. This situation triggers initiation
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of the proofreading pathway. However, if exonuclease complexes also cannot be formed readily,
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then formation of pre-T complexes is favored. Pre-T complexes can be detected by anti-viral
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drugs that mimic pyrophosphosphate, e.g. phosphonoacetic acid (PAA). Pre-T complexes are
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trapped by binding PAA, which hinders further DNA replication [20,31]. The T4 G255S-DNA
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polymerase is PAA-sensitive [18] as expected if polymerase-to-exonuclease switching is
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compromised.
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Normally, pre- and post-T complexes are in rapid equilibrium [30], but formation
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After exonuclease complexes are formed, the terminal residue is removed rapidly [7,32]. T4 DNA polymerase mutants that have amino acid substitutions for conserved active site
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residues are exonuclease and proofreading deficient as observed for other proofreading DNA
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polymerases [33]. The last step in the proofreading pathway is to return the trimmed primer-end
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from the exonuclease to the polymerase active site; this step is rapid for the wild type T4 DNA
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polymerase, but slow for the W213S-DNA polymerase [34].
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Although much is known about proofreading by the T4 and RB69 DNA polymerases,
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less is known about proofreading by other family B DNA polymerases. However, because
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family B DNA polymerases share conserved motifs and overall general structure, similar
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function is expected. For example, the L412M substitution in Motif A in the polymerase active
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site of the T4 DNA polymerase produces a mutator DNA polymerase with enhanced ability to
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extend mismatched primer-templates and the mutant is sensitive to PAA [12,18,20]. We
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expected that both phenotypes would be observed in yeast (Saccharomyces cerevisiae) for the
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L612M-DNA polymerase δ, which has the analogous L-to-M substitution in Motif A. The yeast
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L612M-DNA polymerase δ is an excellent phenocopy of the T4 L412M-DNA polymerase;
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reduced replication fidelity and PAA-sensitivity are observed for both mutant polymerases
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[35,36]. But functional similarities were not observed for T4 DNA polymerase and yeast DNA
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polymerase δ hairpin mutants. The G447S substitution in the tip of the yeast DNA polymerase δ
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hairpin, proposed to be analogous to the G255S substitution in the T4 DNA polymerase (Table
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1), had little effect on replication fidelity [37]. This is surprising because the yeast DNA
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polymerase hairpin resembles the phage T4 and RB69 DNA polymerase hairpins [Fig. 2; 38-
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40]. There is amino acid sequence variation within the hairpins of family B DNA polymerases,
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which could account for different function [37; Table 1], and certainly differences are expected
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for yeast DNA polymerase ε which has only a truncated hairpin [41; Fig. 2]. But the lack of a
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substantial proofreading defect for the G447S-DNA polymerase δ and the absence of a hairpin in
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DNA polymerase ε raise the possibility that not all DNA polymerase hairpin structures are
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required for proofreading.
Genetic selection strategies, modeled on T4 methods, were used to identify proofreading
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deficient yeast DNA polymerase δ mutants. Several yeast mutator DNA polymerases were
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selected with amino acid substitutions in the exonuclease domain that reduced exonuclease
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activity and increased mutation rates, but none of substitutions were in the hairpin region [42].
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Just as mutator DNA polymerases can be selected in phage T4 and yeast, recent large-scale DNA
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sequencing studies reveal that mutator DNA polymerase δ and ε mutants are also selected in
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cancer cells [43-47]; this is because “mutator” DNA polymerases are proposed to provide a pool
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of mutations that are needed to drive development of cancer cells [48]. While several of the
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mutant DNA polymerase and mutants identified in human colon and endometrial tumors
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have amino acid substitutions within the exonuclease domain that reduce 3’ → 5’ exonuclease
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activity [47], no substitutions have been identified in the hairpin region.
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Having failed to find hairpin mutants that affect function of the yeast DNA polymerase δ,
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we constructed hairpin mutants with major structural changes. A hairpin deletion (hpmutant
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was made in which 7 amino acids were deleted (Table 1), as was done for the RB69 DNA
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polymerase hp mutant that lacks 3’→5’ exonuclease activity with duplex, but not single-
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stranded DNA [27] and displays a strong mutator phenotype in vivo [26]. A yeast/T4 chimeric
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DNA polymerase δ was also engineered in which the yeast hairpin was replaced with the T4
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DNA polymerase hairpin (T4hp) (Table 1). An additional hairpin mutant with a single amino
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acid substitution, the G447D-DNA polymerase δ, was constructed because recent studies suggest 7 Page 7 of 48
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that the G447D amino acid substitution curbs the strong mutator phenotype produced by an
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exonuclease-deficient DNA polymerase δ [49]. The major conclusion from these studies is that, unlike the phageT4 and RB69 DNA
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polymerases, yeast DNA polymerase δ does not need the hairpin for proofreading, but appears to
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require the hairpin for optimum DNA replication efficiency. A second conclusion is that MMR
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plays an important role in mediating replication problems caused by DNA polymerase δ mutants
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that is independent of mismatch correction. We discuss the possibility that the two conclusions
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are linked because T4 and RB69 phage do not have MMR and, thus, may have compensated for
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this deficiency by using the hairpin to increase proofreading efficiency. Yeast, on the other
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hand, may depend more on MMR instead of strong proofreading activity and, thus, the hairpin is
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used instead to maintain optimum replication efficiency.
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2. Materials and methods
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2.1. Yeast strains
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The yeast strains used in this study are listed in Supplementary Table S1. All strains are isogenic derivatives of MS71, which is a derivative of CG378/CG379.
2.2. Yeast culture conditions Standard yeast media were used (YPD, YPD plus selective antibiotics, SD medium
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supplemented with essential nutrients or lacking specific nutrients (e.g. uracil, tryptophan,
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histidine, lysine, or SD –Arg, plus canavanine); all media have been described [50]. PAA 8 Page 8 of 48
gradients are described by Li et al. [35]. Gradient plates are made in two steps. First, a pH 5-
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adjusted solution of SD minimal medium made with high grade Noble agar and PAA (Sigma-
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Aldrich) is poured into a square Petri dish and one end is propped up with a pencil. Second, after
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the agar hardens the dish is set flat and a second layer of agar without PAA is poured gently over
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the first layer to preserve the slanted first layer. If the PAA solution contains 4 mg/ml PAA (28
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mM), then the final gradient is from ~0 to 4 mg/ml (~0 to 28 mM) PAA. HU gradient plates
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were prepared as done for PAA gradients, but YPD and Difco agar were used; 0.2M HU was in
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the slanted first layer and the second layer was YPD without HU.
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2.3 Site-directed mutagenesis and mutant construction
Single amino acid substitutions were made using an efficient single mutagenic primer
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method that has been described [50]; details are available by request. The starting point was the
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yeast integrating plasmid, Yip5, that contained the 2.2 kb fragment from the 5’-end of the POL3
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gene (SalI-BamHI fragment) [37]. The DNA polymerase hp mutant was constructed using
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the same Yip5 plasmid. The mutagenic primer for the hp mutant was the following: 5ʹ-P-
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GCAAGAAATTAAAGAGTCTGTGTTCGGTGAAACCAAAAATGTCAATATTGACGG-3ʹ.
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The Yip5-pol3-hpΔ plasmid was sequenced to confirm the deletion. The plasmid was cleaved at
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the unique HpaI restriction endonuclease site and then used to transform competent yeast cells
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that were prepared as described [51]. PCR and DNA sequencing were done to confirm correct
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integration of the plasmid into the chromosomal copy of the POL3 gene and restoration of a
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single gene copy after selection for plasmid pop-out on 5-FOA.
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The chimeric DNA polymerase T4hp mutant was constructed starting with the Yip5 plasmid that contained the longer 2.5 kb fragment from the 5’-end of the POL3 gene (SalI-KpnI
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fragment). A 170 bp fragment containing DNA encoding the yeast hairpin was removed leaving
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PsiI and EagI ends. PCR was used to amplify DNA encoding the T4 hairpin; PsiI and EagI sites
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were engineered into the 5’- and 3’-ends. Plasmid DNA with complimentary PsiI and EagI ends
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was then ligated to the T4 hairpin fragment. The plasmid was sequenced to confirm construction
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of the chimeric yeast/T4 DNA polymerase mutant. The plasmid was cleaved at the unique
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BamHI restriction endonuclease site and transformed into competent yeast cells. PCR and DNA
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sequencing were done to confirm correct integration of the plasmid into the POL3 gene and
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restoration of a single gene copy after selection for plasmid pop-out on 5-FOA.
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2.3 Determining spontaneous mutation rates
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Mutation rates were determined as described previously [35]. The trp1-289 allele reverts by base substitution and the his7-2 and lys2::InsE(A8) alleles revert by +1 and -1 frameshift
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mutations, respectively. Forward mutations to produce resistance to canavanine (CanR) can be
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base substitutions, frameshifts or any mutation that inactivates function (insertion, deletion,
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complex).
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3. Results
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3.1. Replication fidelity by yeast DNA polymerase hairpin mutants with single amino acid
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substitutions: G447S, G447Q, G447D 10 Page 10 of 48
Single amino acid changes in the tip region of the phage T4 DNA polymerase β hairpin,
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e.g. G255S, G255A, Y254G, Y254S, Y254Q, Y254K (14,25; Table 1, Fig. 1B), produce low
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fidelity DNA polymerases that increase base substitution mutations to a level similar to
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exonuclease-deficient DNA polymerase mutants [37], but this was not observed for analogous
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amino acid substitutions in the yeast DNA polymerase δ hairpin. Little change in fidelity was
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observed for the G447S- [37] or G447Q-DNA polymerase δ mutants, but a 7-fold increase in
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-1fs mutations was detected with the Lys+ reporter for the G447D- DNA polymerase δ in the
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absence of MMR and a 3-fold increase in mutations that confer CanR (Table 2).
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3.2 The pol3-G447D strain is sensitive to hydroxyurea (HU) but not to phosphonoacetic acid
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(PAA)
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We tested the possibility that the β hairpin in yeast DNA polymerase δ was needed for
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efficient DNA replication by looking for a slow growth phenotype or HU- or PAA-sensitivity.
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PAA inhibits DNA polymerases that favor formation of pre-T complexes and HU-sensitivity
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identifies “weak” DNA polymerases that have difficulty replicating DNA when deoxynucleoside
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triphosphate (dNTP) pools are reduced due to HU inhibition of ribonucleotide reductase. A slow
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growth phenotype was not observed with rich or minimal media, but cell killing was observed in
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the presence of HU in the absence, but not in the presence of MMR (Fig. 3A). HU induces a
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checkpoint response for wild type yeast that severely slows DNA replication [52], but the
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observed killing by HU for pol3-G447D cells that lack MMR suggests that MMR is needed to
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activate the checkpoint response or to assist in recovery. This observation raises the possibility
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that MMR interacts with the mutant DNA polymerase δ at stalled replication forks. This
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proposal is elaborated further in the Discussion. Although the phage T4 G255S-DNA
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polymerase is PAA sensitive [18], the similar yeast pol3-G447S, Q and D strains were not,
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which indicates functional differences between the yeast and T4 hairpin mutants.
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3.3. The yeast DNA polymerase hp mutant replicates DNA with high fidelity but displays a
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slow growth phenotype
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Seven amino acids from the tip region of the hairpin were deleted as was done to create
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the proofreading-deficient RB69 DNA polymerase hp mutant [27; Table 1]. A strong mutator
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phenotype is observed for the phage RB69 hp strain as expected if proofreading is
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compromised [26]; a similar reduction in replication fidelity was expected for the yeast DNA
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polymerase δ hp (pol3-hp) strain if the yeast hairpin is needed for exonucleolytic proofreading
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activity. The pol3-hp strain replicated DNA almost as accurately as the wild type POL3 strain;
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only a 2-fold increase in the CanR mutation rate was detected in the absence of MMR (Table 2),
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which was due primarily to an increase in -1 fs mutations (Table 3). The small reduction in
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replication fidelity by the DNA polymerase δ hp mutant was less than the weak mutator
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phenotype observed for the pol3-G447D MMR- strain. In comparison, a 20-fold increase in the
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CanR mutation rate is observed for the exonuclease-deficient pol3-5DV strain, and this is in the
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presence of MMR [53]. The pol3-5DV strain is inviable in the absence of MMR because the
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exceptionally high mutation rate produces error castastrophe; see discussion by Herr et al. [54].
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Thus, the yeast DNA polymerase δ hairpin does not appear to function in proofreading because
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replication fidelity is largely retained for the hpmutant.
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A pronounced slow growth phenotype was observed for cells expressing the DNA polymerase δ hp mutant in the absence of MMR, which is shown for the pol3-hp msh6 msh3
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strain (Fig. 4). Yeast cells that lack both Msh6 and Msh3 cannot form MutS α or β complexes
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and, thus, are MMR-deficient [55]. Colonies were significantly smaller for the MMR-deficient
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pol3-hpstrain than for the MMR-proficient pol3-hp strain. The slow growth phenotype for
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the pol3-hp MMR- strain was also observed in liquid culture where the maximal cell titer
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reached only about 2 x 107 cells/ml for the mutant compared to 108 cells/ml for wild type yeast.
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While slow growth could be due to low viability caused by a high mutational burden, only a
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weak mutator phenotype was observed for the pol3-hpΔ MMR- strain (Table 2); instead, an S-
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phase check point appears to be activated as most cells resembled dumbbells as expected if the
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cell cycle has a prolonged S-phase.
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The MRC1 gene was deleted in MMR proficient and deficient pol3-hpstrains to
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determine if an S-phase checkpoint was responsible for the slow growth phenotype (Fig. 4).
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Deletion of the MRC1 gene had little effect on pol3-hpcells in the presence of MMR, but
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deletion partially rescued the slow growth phenotype in the absence of MMR; compare colony
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sizes for pol3-hpMMR- andpol3-hpMMR- mrc1strains. Thus, there are problems with
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DNA replication by the DNA polymerase δ hp mutant, but the problems are masked by MMR.
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DNA replication activity of the hpΔ-DNA polymerase δ was further probed with HU and
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PAA. The pol3- hpstrain, with or without MMR, was not HU-sensitive (Fig. 3A), but PAA-
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resistance was observed for the MMR-proficient pol3- hpstrain (Fig. 3B). A slow growth
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phenotype is observed for wild type yeast cells at high concentrations of PAA after 2 days
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incubation, but colonies were observed across the entire gradient (~0 to 4 mg/ml (28 mM) PAA) 13 Page 13 of 48
by 7 days. In contrast, cells expressing the PAA-sensitive L612M-DNA polymerase δ produced
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only small colonies after 2 days incubation at the lowest PAA concentrations, and more colonies
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were detected after 7 days, but only up to the midway position in the gradient. High PAA
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concentrations killed cells that expressed the L612M-DNA polymerase δ, as observed previously
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[35]. The pol3-hp strain, however, was more PAA-resistant than wild type cells (POL3) as
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colonies were observed at the highest PAA concentrations after just 2 days incubation (Fig. 3B.).
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Even the MMR-deficient pol3-hp strain appeared to be more resistant to PAA despite the slow
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growth phenotype. The PAA-resistance of the hp-DNA polymerase δ indicates reduced ability
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to form pre-T complexes compared to the wild type DNA polymerase δ and, thus, altered DNA
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replication.
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3.4. A strong mutator phenotype for -1fs mutations was observed for the chimeric DNA
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polymerase T4hp mutant in the absence of MMR
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A chimeric yeast-T4 DNA polymerase δ mutant was constructed in which the T4 β
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hairpin replaced the DNA polymerase δ hairpin (pol3-T4hp). The substitution extended from the
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ExoII motif through the hairpin (Table 1). The T4 and yeast DNA polymerase δ hairpins are
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similar (Fig. 2) and, thus, may be expected to substitute for each other. The yeast pol3-T4hp
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strain was viable, but the converse construction, in which the yeast DNA polymerase δ hairpin
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replaced the T4 hairpin in the phage T4 DNA polymerase, was not. Only small increases in
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spontaneous mutation rates were observed for the yeast MMR-proficient pol3-T4hp strain, but
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larger 5- to 20-fold increases in mutation rates were observed for the MMR-deficient pol3-T4hp
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strain compared to the POL3 MMR- strain (Table 2). The CanR mutation spectrum for the 14 Page 14 of 48
MMR-deficient pol3-T4hp strain (pol3-T4hp MMR-) was predominated by -1 fs mutations
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(Table 3) at sites within tracts of 4 to 6 Ts (As on the complementary strand) as observed for the
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POL3 MMR- strain. More than a third of the total CanR mutants sequenced (15/42) for the
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MMR-deficient pol3-T4hp strain were -1fs mutations at a single site within a tract of 6 Ts
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(Supplementary Figure 1S). This “hotspot” mutation site was observed for only 2/20 CanR
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mutants sequenced for the wild type DNA polymerase MMR-deficient strain (POL3 MMR-)
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(Supplementary Figure 1S). The mutation rate for -1 frameshift mutations in the absence of
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MMR for the DNA polymerase T4hp mutant was 5478 x 10-8 ([fraction of -1 fs mutations] x
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[total CanR mutation rate]; 0.90 x 6087) compared to just 278 x 10-8 (0.85 x 363) for the wild
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type DNA polymerase, which is almost a 20-fold increase in the rate for -1fs mutations for the
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T4hp-DNA polymerase δ mutant.
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We were concerned, however, that the large replacement of T4 DNA polymerase
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sequence in the yeast DNA polymerase δ could affect exonuclease activity because non-
327
conserved residues in the ExoII motif in the chimeric pol3-T4hp strain were replaced along with
328
T4 DNA polymerase hairpin residues (Table 1). To test this possibility, the “ttn” chimeric pol3-
329
T4hp strain was constructed in which the yeast ExoII motif was restored to produce the MMR-
330
deficient pol3-T4hp,ttn strain. The ttn substitution reduced the CanR mutation rate about 4-fold
331
(Table 2), which indicates that the ttn substitution contributes to the mutator phenotype observed
332
for the MMR- pol3-T4hp strain. Furthermore, while fs mutations predominated in the CanR
333
spectrum for MMR- pol3-T4hp strain (Table 3, Supplementary Figure 1S), base substitution
334
mutations predominated for the MMR-deficient pol3-T4hp,ttn strain as expected if the mutant
335
DNA polymerase was defective in proofreading misincorporated nucleotides. Note that the
336
phage T4 G255S-DNA polymerase is a strong mutator for base substitutions, but not fs
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15 Page 15 of 48
mutations [37]. However, introducing the T4 ExoII motif into yeast DNA polymerase δ did not
338
create a mutator phenotype; only slightly elevated mutation rates were detected for the MMR-
339
pol3-ieg strain above rates observed for the POL3 MMR- strain (Table 2). Thus, the strong
340
mutator phenotype for fs mutations observed for the pol3-T4hp strain requires the T4 hp and the
341
T4 ExoII motif. If frameshift mutations arise because of DNA polymerase dissociation as
342
proposed by Kunkel et al. [56], then the strong mutator phenotype for -1fs mutations observed
343
for the MMR-deficient pol3-T4hp strain may be caused by increased polymerase dissociation
344
during attempted proofreading, while the increase in base substitution mutations observed for the
345
MMR-deficient pol3-T4hp, ttn strain is due to decreased proofreading.
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3.5. The chimeric DNA polymerase T4hp strain is HU- and PAA-sensitive Like the MMR-deficient pol3-G447D MMR- strain, the MMR-deficient chimeric pol3-
te
347
M
346
T4hp MMR- strain was sensitive to HU (Fig. 3A); thus, the T4hp hairpin-ExoII
350
insertion/replacement may produce a weak DNA polymerase δ. A slow growth phenotype was
351
observed for the chimeric pol3-T4hp MMR- strain, which could indicate slow DNA replication
352
[Fig. 4], but the slow growth may also be due to reduced viability that is caused by error
353
catastrophe. The HU-induced checkpoint may exacerbate the high mutation rate [57] and
354
increase cell death. The pol3-T4hp MMR- strain was also sensitive to PAA, but the pol3-T4hp
355
MMR+ strain appears to be more resistant than the wild type POL3 strain, but not as resistant as
356
the pol3-hpMMR+ strain (Fig. 3). The MMR-deficient pol3-T4hp ttn mutant was not sensitive
357
to HU or to PAA, data not shown.
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358 16 Page 16 of 48
359
361
3.6 T4hp-DNA polymerase δ replication in the absence of Rad27 orExo1 The pol3-T4hp rad27 strain was not viable. Only 3 of 4 spores germinated; cell growth
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362
arrested with 50 to 100 cells. The pol3-hp rad27 mutant, however, was viable.
363
DNA polymerase 3’→5’ exonuclease activity or Rad27 flap endonuclease is required to form
364
ligatable ends at Okazaki fragment junctions and synthetic lethality is observed in the absence of
365
both activities [53,58-60], the T4hp insertion/replacement appears to prevent proofreading. One
366
possibility is that the T4hp-DNA polymerase δ is prone to dissociation in the context of Okazaki
367
fragment junctions.
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368
Since either
The 5’→3’ exonuclease activity of Exo1 may fulfill a similar function as Rad27 in forming ligatable junctions, but at repair gaps. Increased fs mutations are observed for DNA
370
polymerase δ and ε mutants with reduced exonuclease activity in the absence of Exo1 [61]; this
371
is the exo1-dependent mutator (edm) phenotype [62]. An edm phenotype was observed for the
372
pol3-T4hp exo1 strain (Table 2); synergistic increases in +1fs and -1fs mutations were observed
373
for the double mutant. The CanR mutation spectrum for the pol3-T4hp exo1 strain was primarily
374
frameshift mutations (17/20): 10 were +1fs mutations and 7 were –1fs mutations in
375
mononucleotide repeat tracts.
377 378 379
te
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3.7 The T4hp,L612M-DNA polymerase has little proofreading activity If the T4hp-DNA polymerase δ mutant has reduced 3’→5’ exonuclease activity, then the addition of a second amino acid substitution, L612M, that reduces exonuclease activity by 17 Page 17 of 48
380
reducing the opportunity to proofread [12,20], has the potential to create a proofreading-deficient
381
DNA polymerase even though the exonuclease active site is unchanged. This was observed. The pol3-T4hp,L612M strain has a strong mutator phenotype in the presence of MMR
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(Table 4), similar to the strong mutator phenotypes observed for the exonuclease-deficient pol3-
384
01 and pol3-5DV strains [53,58]. Mutation rates could not be determined for the pol3-T4hp,
385
L612M strain in the absence of MMR because the double mutant was not viable as observed for
386
the pol3-01 and pol3-5DV strains. No pol3-T4hp,L612M msh6 mutants were recovered from 31
387
dissected asci. MMR was partially inhibited for the pol3-T4hp,L612M strain by the addition of
388
cadmium (2.5 µM), which inhibits MMR activity [63]. The double mutant was not viable at
389
higher cadmium concentrations likely because of an excessively high mutation burden. The
390
mutation rate for CanR increased 10-fold from 840 x 10-8 to >9000 x 10-8 in the presence of 2.5
391
µM cadmium (Table 4), which is likely at the limit for haploid cell survival [54]. Base
392
substitution and -1 and +1 frameshift mutations were produced for cadmium-treated pol3-T4hp,
393
L612M cells. The pol3-T4hp, L612M strain was also not viable in the absence of Exo1, as
394
observed for the exonuclease-deficient pol3-01 strain [59]; no pol3-T4hp, L612M exo1 mutants
395
were recovered from dissection of 27 asci. The strong mutator phenotype and synthetic lethality
396
of the pol3-T4hp, L612M strain in the absence of Msh6 or Exo1 indicates a severe loss of
397
proofreading activity.
399
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4. Discussion
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18 Page 18 of 48
Experiments presented here with the yeast hpΔ-DNA polymerase δ mutant demonstrate
402
that the yeast β hairpin is not required for proofreading as observed for the phage T4 and RB69
403
DNA polymerase β hairpins. Instead of the strong mutator phenotype that is observed for the
404
phage G255S- and hpΔ-DNA polymerases [14,25,26,37], only a 2-fold increase in frameshift
405
mutations was observed for the pol3-hpΔ strain in the absence of MMR (Tables 2,3), but the
406
slow growth phenotype [Fig. 4] indicates that loss of the hairpin compromises DNA replication.
407
Deletion of MRC1 partially rescued the slow growth phenotype [Fig. 4], which suggests that the
408
intraS-phase checkpoint is activated. While the RB69 DNA polymerase β hairpin acts as a
409
wedge between the template and primer strands in exonuclease complexes (Fig. 1B) the hairpin
410
has also been observed to contact the single-stranded template strand in complexes in which
411
replication is stalled at DNA damage, perhaps in a pre-exonuclease complex [64]. The yeast
412
DNA polymerase δ hairpin has also been shown to contact DNA in polymerase complexes [40];
413
K444 at the tip of the hairpin (Table 1) protrudes into the major groove and the complex is
414
stabilized by interactions with other hairpin residues, F441 and Y446 [40,65]. The position of
415
the yeast β hairpin is consistent with a role in polymerase-to-exonuclease switching [40,65];
416
however, these hairpin residues are missing in the hpΔ-DNA polymerase, but proofreading is
417
largely intact. Thus, in view of the slow growth phenotype observed for the pol3-hpΔ strain in
418
the absence of MMR, the yeast β hairpin appears to play a role in optimizing DNA replication
419
efficiency, perhaps by stabilizing polymerase complexes, rather than in proofreading. If this is
420
the case, then the presence of only a truncated hairpin in DNA polymerase ε that is too short to
421
contact DNA (Fig. 2) may be explained by the presence of the processivity (P) domain, which is
422
proposed to stabilize polymerase complexes [41]. Thus, the β hairpin structures in the T4 and
423
RB69 DNA polymerases may function to stabilize pre-exonuclease and exonuclease complexes,
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19 Page 19 of 48
both sides of polymerase-to-exonuclease switching, but the yeast DNA polymerase δ hairpin
425
may only stabilize polymerase complexes. DNA polymerase ε, on the other hand, does not need
426
the β hairpin and relies instead on the P-domain.
427
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424
Although previous studies indicated little change in replication fidelity for single amino acid substitutions in the tip of the yeast DNA polymerase δ hairpin [37], we constructed the
429
G447D-DNA polymerase δ because the G447D substitution appears to rescue the synthetic
430
lethality of the pol3-01,L612M strain, presumably by reducing the mutation rate and preventing
431
error catastrophe [49]. DNA replication fidelity was determined in the absence of MMR for the
432
double mutant, pol3-G447D,L612M; no change in replication fidelity was observed for the
433
double mutant compared to the po3-L612M strain (data not shown). However, if the G447D
434
substitution increases polymerase dissociation, for example at mismatched primer-ends, this
435
would increase the opportunity for removal of the incorrect nucleotide by another exonuclease.
436
Another possibility is that the mismatched primer-end would be extended by a translesion DNA
437
polymerase, which would be mutagenic if not repaired by MMR, but DNA replication would be
438
able to continue. While HU-sensitivity is observed for the pol3-G447D strain only in the
439
absence of MMR (Fig. 3A), the pol3-G447D,L612M strain is HU-sensitive in the presence of
440
MMR and is killed by very low HU concentrations in the absence of MMR (data not shown).
441
Thus, the G447D substitution in the tip of the hairpin may interfere with DNA interactions
442
needed to stabilize polymerase complexes. HU-sensitivity indicates that the G447D- and
443
G447D,L612M-DNA polymerase δ mutants cannot form stable post-T complexes unless dNTP
444
pools are high.
445 446
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The above discussion points to a role for the β hairpin in stabilizing polymerase complexes. But the slow growth phenotype for the pol3-hpΔ strain was only observed in the 20 Page 20 of 48
absence of MMR; similarly, HU-sensitivity was only observed for the pol3-G447D strain in the
448
absence of MMR. Thus, MMR normally masks replication problems that are caused by the hpΔ
449
and the G447D substitution, but in different ways. While MMR complexes may stabilize
450
polymerase complexes formed with the hpΔ-DNA polymerase δ so that an S-phase checkpoint is
451
not activated, MMR may be needed for pol3-G447D cells to activate an S-phase checkpoint or to
452
assist in recovery since HU kills these cells. We observed previously that MMR is needed to
453
activate the Rad9-dependent checkpoint in PAA-treated pol3-L612M cells [66]. Although
454
further study is needed to reveal the role of MMR in mediating DNA replication problems
455
caused by the hpΔ- and G447D-DNA polymerase δ mutants, MMR is clearly performing a
456
function that is independent of mismatch correction because the mutants have relatively high
457
replication fidelity (Table 2).
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The MMR-deficient pol3-T4hp strain proved to be complex because the strong mutator
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458
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447
phenotype for fs mutations (Tables 2,3) and the HU- and PAA-sensitivity (Fig. 3) depended on a
460
synergistic interaction between T4 DNA polymerase residues in the ExoII motif and in the T4hp.
461
All of the phenotypes observed for the pol3-T4hp MMR- strain disappeared when the yeast ExoII
462
motif was restored in the pol3-T4hp,ttn MMR- strain. A weak mutator phenotype for base
463
substitution mutations, however, was observed for the pol3-T4hp,ttn MMR- strain, which
464
suggests that the T4hp interferes with proofreading, possibly by decreasing polymerase-to-
465
exonuclease switching. These studies demonstrate the evolutionary fine-tuning that has taken
466
place to optimize β hairpin function for the yeast and T4 DNA polymerases.
467
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The pol3-T4hp strain also provided informative comparisons with the pol3-L612M strain.
468
Both mutants display mutator phenotypes in the absence of MMR and the edm-phenotype
469
(Tables 2,3); in addition, both mutant DNA polymerases cannot support DNA replication in the 21 Page 21 of 48
absence of Rad27 [35], but there are important differences. In the presence of MMR, the pol3-
471
L612M strain is PAA-sensitive, but the pol3-T4hp strain is slightly PAA-resistant (Fig. 3B), but
472
in the absence of MMR, PAA kills both mutant strains (Fig. 3B; 35). Another difference is that
473
HU kills pol3-T4hp cells in the absence of MMR (Fig. 3B), but MMR- pol3-L612M cells are
474
only slightly HU-sensitive (data not shown). We reasoned that proofreading was compromised
475
for both mutant DNA polymerases, but in different ways; thus, the double mutant, pol3-
476
T4hp,L612M, has two defects, which together severely reduce the initiation of the proofreading
477
pathway. As observed for exonuclease-deficient DNA polymerases, pol3-T4hp,L612M was not
478
viable in the absence of MMR or Exo1 and a strong mutator phenotype was observed when
479
MMR was partially reduced with cadmium (Table 4). Thus, the inability to perform polymerase-
480
to-exonuclease switching creates exonuclease deficiency as much as inactivation of exonuclease
481
active site residues.
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While family B DNA polymerases share common functions, there is divergence in the
te
482
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470
use of the β hairpin. The T4 DNA polymerase employs the hairpin to optimize proofreading
484
efficiency, and is about 1000-fold more active on double-stranded DNA ([23]; kcat = 15-19 s-1)
485
than the hairpin-less Klenow fragment of Escherichia coli DNA polymerase I ([67]; kcat = 0.001
486
s-1). The importance of the β hairpin for proofreading by the T4 and RB69 DNA polymerases is
487
demonstrated by the high number proofreading-defective mutants that have been identified in the
488
hairpin region (Table 5), and equally striking is the absence of amino acid substitutions in the
489
hairpin regions of yeast and human DNA polymerase δ and ε mutants that create mutator DNA
490
polymerases. In contrast to the absence of hairpin mutants in yeast and human mutator DNA
491
polymerase δ and ε, several amino acid substitutions in the adjacent region produce mutator
492
DNA polymerases, but this region is nearly void of amino acid substitutions for the phage T4
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22 Page 22 of 48
DNA polymerase despite extensive mutational analysis (Table 5). The yeast mutator DNA
494
polymerase δ mutants have reduced exonuclease activity as judged by sensitivity to cadmium
495
[42]. Amino acid substitutions V411L and L424V in human DNA polymerase ε reduce 3’-
496
exonuclease activity [47] and are observed frequently in mutator DNA polymerase ε mutants
497
expressed in cancer cells [44-47]. Interestingly, the prognosis is often good for DNA polymerase
498
ε exonuclease domain mutants [46], which may be due to retention of MMR because reduced
499
viability is observed for exonuclease-deficient mutants in the absence of MMR due to a heavy
500
mutation burden [47]. But another possibility is that if MMR mediates DNA replication
501
problems for DNA polymerase ε mutants as we observe for DNA polymerase δ mutants, then
502
MMR will be retained to maintain efficient DNA replication.
cr
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503
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493
Even though the β hairpin is not essential for proofreading by DNA polymerase δ, other features of the exonuclease active site are conserved and essential. Besides three essential
505
conserved aspartate residues that bind two Mg2+ ions in the catalytic center [33,58], a
506
phenylalanine residue (F123) is observed to intercalate between the terminal bases at the primer-
507
end in exonuclease complexes formed with the RB69 DNA polymerase [39], or is in close
508
proximity to the primer-end (Fig. 1B). Intercalation is likely the normal mode of binding as
509
determined from studies with the fluorescent base analog, 2-aminopurine (2AP) [68]. Highly
510
fluorescent exonuclease complexes are formed with the T4 DNA polymerase with primers
511
labeled at the 3’-end with 2AP because binding unstacks the terminal 2AP and relieves base
512
stacking interactions that quench fluorescence [32]; unstacking is proposed to be mediated by
513
intercalation of F120 in the T4 DNA polymerase or F123 in the RB69 DNA polymerase [68].
514
Fluorescent exonuclease complexes are not observed, however, for the T4 D219A-DNA
515
polymerase [32] and, as shown in Figure 1B, F123 does not intercalate between the terminal
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23 Page 23 of 48
bases for complexes formed with the RB69 D222A-DNA polymerase, which is analogous to the
517
T4 D219A-DNA polymerase. Thus, the D219A/D222A substitution in the ExoII motif prevents
518
phenylalanine intercalation. Intercalation by the conserved phenylalanine residue appears to be
519
important for proofreading because amino acid substitutions for proline residues near the
520
conserved phenylalanine residue, P123L in the T4 DNA polymerase, P286R and P266H in
521
human DNA polymerase ε, and P327R in human DNA polymerase δ were identified by genetic
522
selection for mutator DNA polymerases [11,44-47]; human P286R- and P286H-DNA
523
polymerase mutants are severely exonuclease-deficient [47].
cr
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524
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516
Different family B DNA polymerases appear to have tuned the balance between primer elongation and proofreading to meet the specific needs for DNA replication accuracy and
526
efficiency in different organisms. T4 DNA polymerase proofreading is highly efficient, in large
527
part due to the β hairpin, and this may be needed because T4 phage do not have MMR to repair
528
DNA polymerase errors. Yeast DNA polymerase δ does not appear to use its hairpin for
529
proofreading, but this is acceptable because MMR can correct DNA polymerase replication
530
errors missed by proofreading; however, the yeast polymerase δ hairpin does appear to be needed
531
for optimal DNA replication activity.
533 534
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Conflict of interest statement
The authors declare that there are no conflicts of interest.
535 536
Acknowledgements 24 Page 24 of 48
537
This work was supported with funds from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research. The authors gratefully acknowledge
539
technical assistance from M. Funnel, M. O’Carroll, and A. Radziwon.
ip t
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540
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Supplementary information
Supplementary Table 1S and Figure 1S can be found in the online version, at http://
us
541
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545
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[24] C. Hariharan, L.J. Reha-Krantz, Using 2-aminopurine fluorescence to detect bacteriophage
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T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading
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reactions, Biochemistry 44 (2005) 15674-15684.
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[25] U. Subuddhi, M. Hogg, L.J. Reha-Krantz, Use of 2-aminopurine fluorescence to study the
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role of the β hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA
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polymerases, Biochemistry 47 (2008) 6130-6137.
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[26] A. Trzemecka, D. Plochocka, A. Bebenek, Different behaviors in vivo of mutations in the β
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[27] M. Hogg, P. Aller, W. Konigsberg, S.S. Wallace, S. Doublié, Structural and biochemical
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investigation of the role in proofreading of a β hairpin loop found in the exonuclease domain of a
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replicative DNA polymerase of the B family, J. Biol. Chem. 282 (2007) 1432-1444.
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[28] Aller, P., S. Duclos, S.S. Wallace, S. Doublié, A crystallographic study of the role of
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sequence context in thymine glycol bypass by a replicative DNA polymerase serendipitously
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619
[30] E. Fidalgo da /Silva, S.S. Mandal, L.J. Reha-Krantz, Using 2-aminopurine fluorescence to
620
measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA
621
polymerases, J. Biol. Chem. 277 (2002) 40640-40649.
622
[31] K.E. Zahn, E.P. Tchesnokov, M. Götte, S. Doublié (2011) Phosphonoformic acid inhibits
623
viral replication by trapping the closed form of the DNA polymerase, J. Biol. Chem. 286 (2011)
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25246-25255.
625
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Figure legends
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cr
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Fig.1. (A) Illustration of the T4 DNA polymerase hairpin acting as a wedge to separate the
742
primer and template strands in exonuclease complexes; adapted from [21]. Phage T4 DNA
743
polymerase residues in the tip of the β hairpin, M253, Y254 and G255, correspond to RB69
744
DNA polymerase residues M256, Y257, and G258. (B) A crystallographic snapshot of the
745
hairpin in exonuclease complexes formed with the RB69 DNA polymerase [27]; republished
746
with permission from the authors. The exonuclease active site is identified by residues D114,
747
E116, D222A and D327A. Note that F123 is adjacent to the 3’-terminal adenine nucleotide on
748
the primer strand.
749
Fig.2. Comparison of the hairpin structures in RB69 DNA polymerase (yellow), yeast DNA
750
polymerase δ (cyan), and the atrophied hairpin in yeast DNA polymerase ε (light green, red
751
arrow). The background structure of DNA polymerase ε is in white [41]; republished with
752
permission from the authors.
753
Fig.3. Yeast DNA polymerase δ mutants display different sensitivities to HU and PAA. (A)
754
About 50 cells of each strain were spotted across a gradient of HU from 0 to 0.2M HU. The
755
plates were scanned after 4 days incubation at 30 °C. (B) About 50 cells of each strain were
756
spotted across a gradient of PAA from 0 to 4 mg/ml (0 to 28 mM). The plates were scanned
757
after 2 and 7 days of incubation. The C (control) lane shows cells that were not exposed to PAA.
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35 Page 35 of 48
Fig.4. The yeast hp-DNA polymerase δ mutant displays a slow-growth phenotype. About 100
759
cells were streaked in different sectors; the plate was incubated for 3 days. The pol3-hp strain
760
in the absence of MMR produced the smallest colonies.
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36 Page 36 of 48
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The authors declare that there are no conflicts of interest.
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44 Page 37 of 48
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Graphical Abstract (for review)
Page 38 of 48
cr
β 255 β 212 GWNIEGFDVPYIMNRVKMILGERSMKRFSPI----GRVKSKLIQNMYGSKEIYSID--GVSILDYL
b
M an
T4
us
Table 1 Construction of yeast DNA polymerase hairpin mutants. DNA pol Protein Sequencea
ip t
Table
β 447 β ypolδ G447S,Q,D 400 GYNTTNFDIPYLLNRAKALKVNDFPYFGRLKTVKQEIKESVFSSKAYGTRETKNVNIDGRLQLDLL S,Q,D
400 GYNiegFDIPYLLNRAKALKVNDFPYFGRLKTVKQEIKESVFSSKAYGTRETKNVNIDGRLQLDLL YYYt4tYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY
yhpΔ
400 GYNTTNFDIPYLLNRAKALKVNDFPYFGRLKTVKQEIKESVF-----G--ETKNVNIDGRLQLDLL
chimeric ypolδ T4hpc
400 GYNIEGFDVPYIMNRVKMILGERSMKRFSPI----GRVKSKLIQNMYGSKEIYSID--GRLQLDLL YYYt4t4t4t4t4t4t4t4t4t4t4t4t4t4----t4t4t4t4t4t4t4t4t4t4t--YYYYYYYY
chimeric ypolδ T4hp ttnc
400 GYNTTNFDVPYIMNRVKMILGERSMKRFSPI----GRVKSKLIQNMYGSKEIYSID--GRLQLDLL YYYYYY4t4t4t4t4t4t4t4t4t4t4t4t4----t4t4t4t4t4t4t4t4t4t4t--YYYYYYYY
ce pt
Ac
a
ed
ypolδ ieg
Highly conserved residues are indicated in bold font [37]. The ExoII motif is underlined. Arrows indicate β hairpin strands. c Yeast residues in the chimeric DNA polymerase are indicated by “Y”, T4 residues are indicated by “t4”. b
Page 39 of 48
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Table
Table 2 Spontaneous mutation rates for yeast pol3-hp strains.
Mutation rates per 108 cells His+ Lys+ (+1 fs) (-1 fs)
Strain Properties
Trp (Base subst.)
POL3 MMR+ a, b
2.9 (2.7-3.6)c
1.1 (0.9-1.6)
6.1 (5-10) N=35e
53 (47-72) N=29
us
cr
+
CanR (Base subst., fs, complex) 31 (27-39)
148 (104-300) N=15
363 (260-538) N=35
20
nd
350
23 (16-38) N=15
nd
333(133-384) N=15
42 (34-71) N=15
1074 (586-1605) N=15
936 (633-1461) N=15
1.2 (0.8-1.7) N=19
0.4 (