High-resolution analysis of protein, and DNA conformational changes during DNA polymerization, established relationships between the enzymatic function and conformational dynamics of individual domains for a DNA polymerase.

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Sulfolobus solfataricus P2 is an aerobic crenarchaeon which grows optimally at 80 degrees C and pH 2-4. This organism encodes a B-family DNA polymerase, DNA polymerase B1 (PolB1), which faithfully replicates its genome of 3 million base pairs. Using pre-steady-state kinetic methods, we estimated the fidelity of PolB1 to be in the range of 10(-6) to 10(-8), or one error per 10(6) to 10(8) nucleotide incorporations in vivo. To discern how the polymerase and 3' --> 5' exonuclease activities contribute to the high fidelity of PolB1, an exonuclease-deficient mutant of PolB1 was constructed by mutating three conserved residues at the exonuclease active site. The base substitution fidelity of this mutant was kinetically measured to be in the range of 10(-4) to 10(-6) at 37 degrees C and pH 7.5. PolB1 exhibited high fidelity due to large differences in both ground-state nucleotide binding affinity and nucleotide incorporation rates between correct and incorrect nucleotides. The kinetic partitioning between the slow mismatch extension catalyzed by the polymerase activity and the fast mismatch excision catalyzed by the 3' --> 5' exonuclease activity further lowers the error frequency of PolB1 by 14-fold. Furthermore, the base substitution error frequency of the exonuclease-deficient PolB1 increased by 5-fold as the reaction temperature increased. Interestingly, the fidelity of the exonuclease-deficient PolB1 mutant increased by 36-fold when the buffer pH was lowered from 8.5 to 6.0. A kinetic basis for these temperature and pH changes altering the fidelity of PolB1 was established. The faithful replication of genomic DNA catalyzed by PolB1 is discussed.

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Active deep-sea hydrothermal vents harbor abundant thermophilic and hyperthermophilic microorganisms. However, microbial communities in inactive hydrothermal vents have not been well documented. Here, we investigated bacterial and archaeal communities in the two deep-sea sediments (named as TVG4 and TVG11) collected from inactive hydrothermal vents in the Southwest India Ridge using the high-throughput sequencing technology of Illumina MiSeq2500 platform. Based on the V4 region of 16S rRNA gene, sequence analysis showed that bacterial communities in the two samples were dominated by Proteobacteria, followed by Bacteroidetes, Actinobacteria and Firmicutes. Furthermore, archaeal communities in the two samples were dominated by Thaumarchaeota and Euryarchaeota. Comparative analysis showed that (i) TVG4 displayed the higher bacterial richness and lower archaeal richness than TVG11; (ii) the two samples had more divergence in archaeal communities than bacterial communities. Bacteria and archaea that are potentially associated with nitrogen, sulfur metal and methane cycling were detected in the two samples. Overall, we first provided a comparative picture of bacterial and archaeal communities and revealed their potentially ecological roles in the deep-sea environments of inactive hydrothermal vents in the Southwest Indian Ridge, augmenting microbial communities in inactive hydrothermal vents.