Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen.
Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.
Kedar
Padmini S
PS
Laboratory of Structural Biology, NIEHS/National Institutes of Health, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
Kim
Soon-Jong
SJ
Robertson
Anthony
A
Hou
Esther
E
Prasad
Rajendra
R
Horton
Julie K
JK
Wilson
Samuel H
SH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D019951:DNA Polymerase beta