DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region.
DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.
Shimazaki
Noriko
N
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan. snoriko@rs.noda.tus.ac.jp
Yazaki
Takaya
T
Kubota
Takashi
T
Sato
Asami
A
Nakamura
Ayako
A
Kurei
Shunsuke
S
Toji
Shingo
S
Tamai
Katsuyuki
K
Koiwai
Osamu
O
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D018809:Proliferating Cell Nuclear Antigen; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C415367:DNA polymerase beta2; D004253:DNA Nucleotidylexotransferase; D019951:DNA Polymerase beta