Abstract
Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. Mac-RFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.
Highlights
Polymerase onto the template [2]
Construction of Plasmids for Overproduction of replication factor C (RFC) Proteins—During annotation of the genome sequence of the mesophilic archaeon M. acetivorans (Mac), we discovered two genes coding for two different RFCS subunits designated MacRFCS1 and MacRFCS2 and another gene coding for one RFCL subunit (MacRFCL)
Two genes coding for two RFCS subunits and one coding for one RFCL subunit were identified in the genome sequence of M. acetivorans during genome annotation
Summary
Polymerase onto the template [2]. The critical functions of sliding clamps and clamp loaders are underscored by their conservation across the three domains of life and in bacteriophage such as T4 phage [3]. This clamp loader type (composed of two RFCS subunits and one RFCL subunit) may represent a critical link in the evolution of complex clamp loaders from simple forms in the archaeal/eukaryotic sister lineages. They are unique among the species of Archaea in forming complex multicellular structures [21] The discovery of this new form of RFC in these organisms is of major importance, as further biochemical and genetic analyses may provide us with critical insights into cellular developments that might have led to a requirement for more complex clamp loaders from the competent but simple ones found in the archaeal/eukaryotic sister lineages
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