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High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases.

Nucleic Acids Res.. 2016-10; 
KM Schermerhorn, N Tanner, Z Kelman, AF Gardner.
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Codon Optimization ... The 9°N MCM2 and MCM3 genes (lacking inteins) were codon optimized for expression in E. coli, constructed synthetically (Genscript, Piscataway, NJ) and cloned into pAII17 plasmid vector (19) cleaved with NdeI and BamHI to produce plasmids pERN (9°N MCM2) and pEUK ... Get A Quote

摘要

The minichromosome maintenance (MCM) complex is the replicative helicase responsible for unwinding DNA during archaeal and eukaryal genome replication. To mimic long helicase events in the cell, a high-temperature single-molecule assay was designed to quantitatively measure long-range DNA unwinding of individual DNA helicases from the archaeons Methanothermobacter thermautotrophicus (Mth) and Thermococcus sp. 9°N (9°N). Mth encodes a single MCM homolog while 9°N encodes three helicases. 9°N MCM3, the proposed replicative helicase, unwinds DNA at a faster rate compared to 9°N MCM2 and to Mth MCM. However, all three MCM proteins have similar processivities. The implications of these observations for DNA repl... More

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