Abstract:DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C abov… Show more
“…The little finger domain and associated linker in particular seem to be most important for stable binding to DNA. On the other hand, the binding affinity of Dpo1 to DNA is more favorable at higher temperature, consistent with formation of a tight closed conformation on DNA, resulting in greater DNA stabilizing ability/annealing noted previously (74). …”
Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism, Sulfolobus solfataricus (Sso), contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein crosslinking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding by Dpo1 monomer is favored over Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration and temperature dependent discrimination of binding undamaged DNA templates at physiological temperatures.
“…The little finger domain and associated linker in particular seem to be most important for stable binding to DNA. On the other hand, the binding affinity of Dpo1 to DNA is more favorable at higher temperature, consistent with formation of a tight closed conformation on DNA, resulting in greater DNA stabilizing ability/annealing noted previously (74). …”
Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism, Sulfolobus solfataricus (Sso), contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein crosslinking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding by Dpo1 monomer is favored over Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration and temperature dependent discrimination of binding undamaged DNA templates at physiological temperatures.
“…This is especially noteworthy in light of our results with homopolymeric substrates in which most incorporation is with nucleotides complimentary to the substrate. Very similar results have been observed for DNA polymerase I from Sulfolobus solfataricus (a B-family DNA polymerase), in which it was proposed that this polymerase is able to add nontemplated bases to the end of a single-stranded template as well as stabilize poorly annealed ends to stimulate template-dependent synthesis (43). Interestingly, the terminal transferase activity proposed for that polymerase was only observed on oligonucleotides that were a minimum of 10–15 nucleotides in length, which is the minimum required length for our activity with POLQ.…”
The biological role of human DNA polymerase θ (POLQ) is not yet clearly defined, but it has been proposed to participate in several cellular processes based on its translesion synthesis capabilities. POLQ is a low-fidelity polymerase capable of efficient bypass of blocking lesions such as abasic sites and thymine glycols as well as extension of mismatched primer termini. Here, we show that POLQ possesses a DNA polymerase activity that appears to be template independent and allows efficient extension of single-stranded DNA as well as duplex DNA with either protruding or multiply mismatched 3′-OH termini. We hypothesize that this DNA synthesis activity is related to the proposed role for POLQ in the repair or tolerance of double-strand breaks.
“…As shown in Figure 1B, PolB1 synthesized products much longer than 72 nt on the circular template, indicating that the polymerase displaced the primer P36 and the newly synthesized strand after gap-filling synthesis. The long products apparently did not result from terminal transfer by PolB1, which was reported to possess an efficient terminal transferase activity (40) because a product obtained on a linear template L72 was 72 nt in length under our assay conditions in a control experiment (Figure 1A and B). Figure 1.DNA strand displacement by PolB1.…”
Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.
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