14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif.
Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.
Department of Science, Systems and Models, Roskilde University, Denmark.
Sofie Dabros
SD
Andersen
Guido
G
Keijzers
Emmanouil
E
Rampakakis
Kim
K
Engels
Patricia
P
Luhn
Mahmoud
M
El-Shemerly
Finn Cilius
FC
Nielsen
Yuhong
Y
Du
Alfred
A
May
Vilhelm A
VA
Bohr
Stefano
S
Ferrari
Maria
M
Zannis-Hadjopoulos
Haian
H
Fu
Lene Juel
LJ
Rasmussen
D048948:14-3-3 Proteins / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D059447:Cell Cycle Checkpoints; D002467:Cell Nucleus / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D048948:14-3-3 Proteins; D050505:Mutant Proteins; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; D045643:DNA Repair Enzymes