Crystal Structures of an NH<sub>2</sub>-Terminal Fragment of T4 DNA Polymerase and Its Complexes with Single-Stranded DNA and with Divalent Metal Ions

The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3 3 5 exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G ؉ C-or A ؉ T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.Exonucleolytic proofreading by DNA polymerases is an important component of high fidelity DNA replication. Nucleotide insertion errors occur at a frequency of 10 Ϫ3 to 10 Ϫ5 , but the DNA polymerase-associated 3Ј 3 5Ј-exonuclease activity further reduces replication errors by 100-fold or more (reviewed in Ref. 1). Different steps of the proofreading reaction have been revealed by mutational analysis of the bacteriophage T4 DNA polymerase. Mutant T4 DNA polymerases deficient in 3Ј 3 5Ј-exonuclease activity exhibit a strong mutator phenotype that can be used to select for mutant DNA polymerases (2-4). The loss of the 3Ј 3 5Ј-exonuclease activity by mutation can be achieved by preventing distinct steps in the proofreading process. The hydrolysis reaction may be prevented if residues required for catalysis are substituted with other amino acids. For example, the substitution of Ala residues for Asp residues that bind essential Mg 2ϩ ions in the exonuclease active center reduces the exonuclease activity to barely detectable levels (5-7). Another reaction step in proofreading that may be affected by mutation is the translocation of the primer end of the DNA between the spatially distinct polymerase and exonuclease active centers. Identification of several "active-site switching mutants" has been described recently (8, 9). DNA polymerase proofreading is a dynamic process; if "switching" to the exonuclease active site is reduced by mutation, there is less opportunity for proofreading and a mutator phenotype is produced (9).Another proofreading reaction step that may be probed by mutational analysis of T4 DNA polymerase is strand separation. The 3Ј 3 5Ј-exonuclease activity o...

supporting

confidence: 90%

mentioning

confidence: 99%

Using 2-Aminopurine Fluorescence and Mutational Analysis to Demonstrate an Active Role of Bacteriophage T4 DNA Polymerase in Strand Separation Required for 3′→ 5′-Exonuclease Activity

Marquez

Reha-Krantz

1996

“…Structural and mutagenesis studies of DNA pol I suggest a role for this conserved Tyr at the active site (31,32). For T4 DNA pol mutations at this Tyr affect both the polymerase and the exonuclease activities (35,39). An alternative Exo III motif, named Exo III⑀, has been proposed based on mutagenesis studies of the exonuclease domain of Bacillus subtilis DNA pol III (40).…”

Section: Resultsmentioning

confidence: 99%

“…Structural studies of DNA pol I indicate that the Tyr in the YXXXD sequence of the Exo III motif interacts with a phosphate oxygen of the 3Ј-terminal nucleotide in the DNA substrate (31). In contrast, the equivalent Tyr in the Exo III motif of bacteriophage T4 DNA pol points away from the active site (35). Currently, structural data of exonucleases containing the HXAXXD sequence in the Exo III motif are not available to access the roles of the amino acids within this sequence.…”

Section: Table I Expression Of Trex Proteins In E Colimentioning

confidence: 99%

Identification and Expression of the TREX1 and TREX2 cDNA Sequences Encoding Mammalian 3′→5′ Exonucleases

Mazur

Perrino²

1999

249

216

The 335 exonucleases catalyze the excision of nucleoside monophosphates from the 3 termini of DNA. We have identified the cDNA sequences encoding two 335 exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 335 exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3 terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.The multistep processes of DNA replication, repair, and recombination in human cells often require the excision of nucleotides from the DNA 3Ј termini. For each cell division 4 billion nucleotides must be correctly replicated. The polymerization of incorrect or structurally modified nucleotides into DNA generates the 3Ј termini that block chain elongation by the DNA polymerases. Oxidative damage to DNA can result in fragmented nucleotides at the 3Ј termini that can not be elongated by DNA polymerases. Genetic recombination and mismatch repair pathways can require the removal of normal nucleotides from the 3Ј termini of DNA chains. Enzymes containing 3Ј35Ј exonuclease activity remove these mismatched, modified, fragmented, and normal nucleotides to generate the appropriate 3Ј termini for subsequent steps in the DNA metabolic pathways.Several 3Ј35Ј exonucleases have been described from a variety of animal cells (1-5). These exonucleases demonstrate similar biochemical properties, but the relationships between these enzymes are not known. Also, there are 3Ј35Ј exonucleases contained in the structural domains of mammalian DNA pols 1 ␦ (6), ⑀ (7), and ␥ (8). These proofreading 3Ј35Ј exonucleases excise incorrectly polymerized nucleotides during DNA synthesis. Thus, a variety of 3Ј35Ј exonucleases are present in mammalian cells. These exonucleases might function in multiple pathways to generate 3Ј termini that support further steps such as polymerization or ligation.Excision of incorrectly polymerized nucleotides by proofreading 3Ј35Ј exonucleases is an important mechanism to minimize errors during DNA synthesis. The polymerase-associated proofreading exonuclease was ...

“…Structural studies of a T4 DNA polymerase fragment showed that the glycine residue at position 255 resides at the tip of a ␤ hairpin loop within the exonuclease domain of the protein (11), but so far there is no structure of full-length T4 DNA polymerase in complex with DNA. The homologous polymerase from bacteriophage RB69, gp43, which shares 61% sequence identity with the T4 polymerase (12), has been crystallized in an apoform (13) and with both normal and damaged DNA (14 -17).…”

mentioning

confidence: 99%

Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family

Hogg

Aller

Konigsberg

et al. 2007

120

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a ␤ hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the ␤ hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the ␤ hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 Å crystal structure of the ␤ hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the ␤ hairpin loop.