Elucidation of an Archaeal Replication Protein Network to Generate Enhanced PCR Enzymes

Abstract

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.For all forms of life, the process of DNA-replication is essential in the propagation of genetic information. A complex multiprotein machinery including DNA polymerases, processivity factors, proof-reading, repair, and regulatory activities (1-3) has evolved to handle the tasks associated with this process. The importance of replication is demonstrated by the fact that its central features are highly conserved among all cellular organisms, while the protein sequences of some of the factors are not. This is exemplified by the processivity factors of DNA polymerases. Processivity is defined as the number of polymerization events during a single contact between polymerase and template. To prevent dissociation off the template DNA, many polymerases are bound by a protein that encircles the DNA in a ring-like structure. Together with the polymerase, the ring appears to move along the DNA as replication proceeds. Such "sliding clamps" exist both for eubacteria (the ␤-subunit of the polymerase) and for eukaryotes (PCNA), 1 and the structures of these proteins are almost superimposable (4). However, the proteins are highly deviant in primary sequence. This situation of sequence divergence and functional conservation is also evident for the proteins responsible for loading the sliding clamp onto the DNA. Whereas in eukaryotes the clamp loader is a heteropentameric complex called RFC (2, 5), the eubacterial clamp loader is represented by the pentameric so called ␥-complex (6, 7). Until recently very little was known about replication in the third domain of life, ...

Generalized walks-based centrality measures for complex biological networks

“…These PPI networks correspond to Archaeglobus fulgidus (Motz et al, 2002), Kaposi sarcoma-associated herpes virus (KSHV) (Uetz et al, 2006), varicella-zoster virus (VZV) (Uetz et al, 2006), Bacillus subtilis (Noirot and NoirotGros, 2004;Hoebeke et al, 2001), Escherichia coli (Butland et al, 2005)), malaria parasite Plasmodium falciparum (LaCount et al, 2005), the worm Caenohabditis elegans (Li et al, 2004),…”

Subunit Interaction and Regulation of Activity through Terminal Domains of the Family D DNA Polymerase from Pyrococcus horikoshii

“…analyzed by in vivo immunoprecipitation assays, in vitro pulldown assays in P. furiosus (26), and yeast two-hybrid assays in A. fulgidus (27). Proliferating cell nuclear antigen was found to stimulate the polymerization activity of PolD of P. furiosus (26).…”

The diverse spectrum of sliding clamp interacting proteins

“…These interactions lead to a model of a sequential recruitment of these proteins to the lagging strand, via their interactions with PCNA, during its maturation to a continuous duplex DNA [25,26]. To date, one of the proteins needed for the process that has not been shown to interact with PCNA is RNase H. The observation that the archaeal RNase H protein binds PCNA (Table 2) [20] suggests that the eukaryotic protein may also bind PCNA. It will be of interest to investigate whether such an interaction exists.…”

“…This approach has the potential to identify additional sliding clamp interacting proteins in the context of large complexes. Other approaches, such as the two-hybrid screen [20], may also lead to new clamp interacting proteins.…”