DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3′ end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3′–5′ exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Forster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site into bulk solution and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.
The integrity of the genetic information is dependent on the fidelity of DNA replication and several DNA repair processes. Among these repair systems, DNA mismatch repair (MMR) is responsible for correcting base-base mismatches and small nucleotide insertion/deletion (IDL) mispairs that arise from polymerase misincorporation, elevating fidelity of replication 50-1000 fold. MMR is initiated when MutS binds to mismatched bases on dsDNA. The communication between the mismatch site and a distal strand discrimination signal is required for removing the mismatch from the newly synthesized strand. MutS-MutL-heteroduplex ternary complex is thought to play an key role in the coupling of these two sites on DNA. We used single molecule fluorescence resonance energy transfer (smFRET) to characterize conformational changes in this ternary complex through the process of mismatch recognition, MutS-MutL interaction and large MutS-MutL assembly formation on DNA. We found that the sliding clamp formation of MutS is inhibited by MutL interaction. The initial MutS-MutL complex stays at the mismatch site and recruits more MutS and MutL to form a large protein assembly. The structural information revealed by our single molecule measurements provides constraints for modeling the mechanism of MMR in the initiation stage.
The accumulation dynamics of polyprenols in leaves of 1-, 2-, and 3-year-old Althaea armeniaca growing in Tashkent were studied according to vegetative phase. Optimal conditions for isolating the polyprenols were determined. It has been shown that the content of polyprenols was highest during fruiting in the second year of growth.
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