The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.
A glycine-rich motif described as being involved in human polymerase delta proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the thermal stability of all mutants was reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region. These studies suggest that the (G)-PYF box motif mediates intersubunit interactions and that it may be crucial for the thermostability of PabPol D.
Castrec
Benoît
B
Université de Bretagne Occidentale, UMR 6197, Laboratoire de Microbiologie et Environnements Extrêmes, BP 70, F-29280 Plouzané, France.
Laurent
Sébastien
S
Henneke
Ghislaine
G
Flament
Didier
D
Raffin
Jean-Paul
JP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D005998:Glycine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010078:Oxazines; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D043903:Pyrococcus abyssi / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D005456:Fluorescent Dyes; D010078:Oxazines; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D004258:DNA Polymerase III; C044808:nile red; D005998:Glycine