A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen.
Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase-PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.
Kiyonari
Shinichi
S
Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka, 812-8581, Japan.
Kamigochi
Toru
T
Ishino
Yoshizumi
Y
D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D005971:Glutamates; D018809:Proliferating Cell Nuclear Antigen; D010649:Phenylalanine; D011189:Potassium Chloride; D011088:DNA Ligases; D000072481:DNA Ligase ATP