The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication.

Abstract

DNA replication in mammalian cells occurs in discrete nuclear foci called ‘replication factories’. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non‐S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)‐DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N‐terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub‐nuclear compartmentalization.

Specific Function of DNA Ligase I in Simian Virus 40 DNA Replication by Human Cell-free Extracts Is Mediated by the Amino-terminal Non-catalytic Domain

“…The inhibitory effect of the amino-terminal domain of DNA ligase I alone and the requirement for this domain to achieve efficient complementation of 46BR.1G1 extracts support the notion that the function of DNA ligase I during replication is mediated by specific binding of this amino-terminal non-catalytic domain to other replication protein(s). This concurs with in vivo studies showing that the first 115 amino acids of DNA ligase I are required for colocalization of the enzyme with bromodeoxyuridine at replication foci (10). As the aminoterminal fragment of DNA ligase I is not as potent an inhibitor of DNA replication as the full-length protein, folding of this region of the protein may be somewhat altered in the absence of the carboxyl-terminal catalytic domain.…”

“…Although this amino-terminal region is not required for activity of DNA ligase I in standard in vitro DNA joining assays, it is essential in vivo to counteract the lethal effect of knocking out DNA ligase I in mouse embryonic stem cells by the ectopic expression of DNA ligase I (9). A functional role for the amino-terminal portion of DNA ligase I during DNA replication has not been directly demonstrated, but this region may serve to localize the protein to sites of DNA replication by specific contacts with other replication factors (10).…”

The Dispersal of Replication Proteins after Etoposide Treatment Requires the Cooperation of Nbs1 with the Ataxia Telangiectasia Rad3-Related/Chk1 Pathway

“…Nuclei were stained with 0.1 Ag/mL 4,6V-diamidino-2-phenylindole (DAPI; Sigma). To detect sites of replicative DNA synthesis, cells were grown in 100 Amol/L BrdUrd (Sigma) for 5 minutes before fixation and processed as previously described (25). Conventional epifluorescence microscopy was done with Optical Microscope Olympus IX71 equipped with a 63Â objective.…”

Eukaryotic DNA Ligases: Structural and Functional Insights

“…This N-terminal region of human DNA ligase I was not present in the catalytic fragment that was crystallized with nicked DNA (26), and it is not required for complementation of yeast cdc9 mutant strains by ligase I. Notably, this region contains sequences that are required for nuclear localization and targeting to replication factories (40)(41)(42), and it is involved in protein-protein interactions described below.…”