DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.
DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.
Vago
Riccardo
R
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy.
Leva
Valentina
V
Biamonti
Giuseppe
G
Montecucco
Alessandra
A
D000906:Antibodies / Q000032:analysis; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004249:DNA Damage / Q000235:genetics / Q000502:physiology; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods
D000906:Antibodies; D018797:Cell Cycle Proteins; C000607892:LIG1 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP