Function and Structure Relationships in DNA Polymerases

Joyce, C.

doi:10.1146/annurev.biochem.63.1.777

Cited by 95 publications

(137 citation statements)

References 89 publications

(200 reference statements)

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“…The wild-type pol/exo balance Most DNA-dependent DNA polymerases display at least two catalytic activities: DNA polymerization and 3'-5' exonucleolysis. Based on structural and functional studies of E.coli DNA polymerase I, and other model enzymes such as herpes simplex virus (HSV), T4 and o29 DNA polymerases, it has been predicted that all these enzymes contain two independent and relatively distant active sites, forming part of two distinct structural domains (reviewed by Joyce and Steitz, 1994). However, in spite of this physical separation, these two opposite activities, synthetic and degradative, must be coordinated efficiently in order to achieve a productive and accurate replication reaction.…”

Section: Discussionmentioning

confidence: 99%

“…Many DNA-dependent DNA polymerases have been shown to contain at least two catalytic activities, DNA polymerization and 3'-5' exonucleolysis, physically separated into two structural domains (reviewed by Joyce and Steitz, 1994). As was proposed for Escherichia coli DNA polymerase I (Pol I), this separation provides the structural framework for a fine-tuned coupling of these two opposite activities, synthetic and degradative, in order to achieve a productive and faithful DNA synthesis (Joyce and Steitz, 1987).…”

Section: Introductionmentioning

confidence: 99%

“…The proposal that all DNA-dependent DNA polymerases may be variations of a common structure is supported by the comparison of the protein sequences and the similarities found in the three-dimensional structure of Klenow, human immunodeficiency virus (HIV) reverse transcriptase and rat DNA polymerase 3 (reviewed by Joyce and Steitz, 1994;Sawaya et al, 1994). However, the published data only support this extrapolation in the portion corresponding to the polymerization active site, and in that corresponding to the exonuclease active site of proofreading enzymes.…”

Section: Introductionmentioning

confidence: 99%

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A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

et al. 1996

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The functional significance of the conserved motif ‘YxGG/A’, located between the 3′‐5′ exonuclease and polymerization domains of eukaryotic‐type DNA polymerases, has been studied by site‐directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3′‐5′ exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the ‘YxGG/A’ motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3′‐5′ exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N‐terminal and C‐terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross‐talk between synthesis and degradation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

et al. 1996

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“…Interestingly, in the presence of its preferred activator (Mn 2ϩ ), Pol μ behaves as a strong mutator, lacking any base discrimination during nucleotide insertion. Structural studies demonstrated that two metal ions, coordinated by a triad of carboxylate residues, are required at the active site of polymerases (for a review, see Joyce and Steitz, 1994). Although Mg 2ϩ -activated catalysis has been the form studied most extensively, other metal ions such as Mn 2ϩ , Co 2ϩ , Ni 2ϩ or Zn 2ϩ can also serve as DNA polymerase activators in vitro, although it is unknown at present which metal or metals are used in vivo.…”

Section: Pol μ a Mutator Dna Polymerasementioning

confidence: 99%

DNA polymerase mu (Pol micro), homologous to TdT, could act as a DNA mutator in eukaryotic cells

2000

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A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.

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“…Despite the obvious differences between the processes of replication and tran-proportion of nascent molecules abort at positions 4 and 5, not progressing into the conformational change scription, the overall three-dimensional structure of T7 RNA polymerase ) is similar to that described here as the transition preceding the elongation mode. This suggests that a proper strand displacement of the polymerization domain of DNA-dependent DNA polymerases (see reviews by Joyce and Steitz, 1994; capacity may be required to facilitate transition, perhaps by the establishment of the initial interactions with the Sousa, 1996), suggesting that the mechanistic problems associated with 'de novo' nucleic acid synthesis could be nascent displaced strand. This could also be related to the fact that T7 RNA polymerase, when engaged in abortive similar in the case of T7 RNA polymerase and φ29 DNA polymerase.…”

Section: And Also In Human Adenovirusmentioning

confidence: 99%

Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase

Méndez¹,

Blanco

Salas

1997

The EMBO Journal

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Phage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide-long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6-9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of phi29 TP-DNA particularly under conditions that decrease the strand-displacement capacity of phi29 DNA polymerase.

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Function and Structure Relationships in DNA Polymerases

Cited by 95 publications

References 89 publications

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

DNA polymerase mu (Pol micro), homologous to TdT, could act as a DNA mutator in eukaryotic cells

Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase

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