title: High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases.
abstract: 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 replication in archaea and the differences and similarities among helicases from different microorganisms are discussed. Development of the high-temperature single-molecule assay establishes a system to comprehensively study thermophilic replisomes and evolutionary links between archaeal, eukaryal, and bacterial replication systems.
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA.
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Schermerhorn
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA tanner@neb.com.
authors.forename: Nathan
authors.initials: N
authors.lastname: Tanner
authors.affiliation: Biomolecular Labeling Laboratory, National Institute of Standards and Technology and Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Kelman
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA gardner@neb.com.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Gardner
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D019641:DNA, Archaeal / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D017013:Methanobacteriaceae / Q000201:enzymology; D011993:Recombinant Fusion Proteins; D013696:Temperature; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D011993:Recombinant Fusion Proteins; D004265:DNA Helicases