Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.
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.
Motz
Michael
M
Exploratory Research, LION Bioscience Ktiengesellschaft, D-69120 Heidelberg, Germany.
Kober
Ingo
I
Girardot
Charles
C
Loeser
Eva
E
Bauer
Ulrike
U
Albers
Michael
M
Moeckel
Gerd
G
Minch
Eric
E
Voss
Hartmut
H
Kilger
Christian
C
Koegl
Manfred
M
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D019609:Archaeoglobus / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D019641:DNA, Archaeal / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding
D019843:Archaeal Proteins; D017931:DNA Primers; D019641:DNA, Archaeal; D018809:Proliferating Cell Nuclear Antigen; D004259:DNA-Directed DNA Polymerase