Abstract
The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.
Highlights
In all forms of life, successful chromosomal DNA replication requires efficient unwinding of the DNA double helix at the replication forks, a reaction catalyzed by the replicative DNA helicase
In eukaryotes the replicative helicase is the CMG complex, a tripartite molecular machine composed of Cdc45, MCM and GINS
In this report we describe the first results of reverse genetic analysis of archaeal MCM function in vivo, using the haloarchaeal organism Haloferax volcanii as a model system
Summary
In all forms of life, successful chromosomal DNA replication requires efficient unwinding of the DNA double helix at the replication forks, a reaction catalyzed by the replicative DNA helicase. In eukaryotes the replicative helicase is the CMG complex, a tripartite molecular machine composed of Cdc, MCM and GINS (reviewed by Onesti and MacNeill, 2013). MCM is a ring-shaped hexamer composed of six related but non-identical subunits, each of which is a member of the AAA+ (ATPases associated with diverse cellular activities) protein superfamily (Duderstadt and Berger, 2008). Cdc and GINS assemble at the G1-S boundary to form the CMG, activation of which involves MCM subunit phosphorylation by DDK (Dbf4-dependent protein kinase). Individual CMG complexes move with the replication forks from origin to inter-origin sequences
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