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

Abstract

The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the fulllength protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.Several distinct DNA ligases have been identified in mammalian cells, DNA ligase I being a major activity in proliferating cells (1, 2). Cytostaining experiments with antibodies against DNA ligase I showed that the enzyme is specifically localized in the nucleus with the same granular staining pattern as DNA polymerase ␣, implicating DNA ligase I in DNA replication (3). DNA ligase I (in conjunction with DNA polymerase ␣, ␦, or ⑀; RNase H1; and the 5Ј-nuclease DNase IV/ FEN-1) is able to complete lagging strand DNA replication in vitro on a synthetic DNA substrate (4), and DNA ligase I activity also functions to generate closed circular DNA during SV40 DNA replication reconstituted with SV40 large T antigen and purified mammalian proteins (5, 6).The human DNA ligase I cDNA encodes a 102-kDa polypeptide (7). A 78-kDa carboxyl-terminal domain shows significant amino acid sequence homology to the CDC9 and cdc17 ϩ gene products of Saccharomyces cerevisiae and Schizosaccharomyces pombe as well as to the human DNA ligase III and IV cDNAs. This domain is catalytically active in vitro in the absence of the amino-terminal region and is able to complement a conditional/ lethal DNA ligase mutant of Escherichia coli (8). The 24-kDa amino-terminal portion of human DNA ligase I is a proteasesensitive hydrophilic region that has no counterpart in other mammalian DNA ligases or the yeast DNA ligases. 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 locali...

CTG/CAG Repeat Instability Is Modulated by the Levels of Human DNA Ligase I and Its Interaction with Proliferating Cell Nuclear Antigen

“…In agreement with published studies, DNA replication efficiency was dependent upon having enough ligation activity and the ability of hLigI to interact with PCNA (16 -18, 33, 57-59). Expression of wild type hLigI mostly corrected the replication defect of hLigI-deficient 46BRLigI m/m extract (17,18,40). These results are also consistent with PCNA coordinating the action of hLigI during Okazaki fragment processing and ligation and the essential role of PCNA in efficient fork progression (17,40).…”

“…Expression of wild type hLigI mostly corrected the replication defect of hLigI-deficient 46BRLigI m/m extract (17,18,40). These results are also consistent with PCNA coordinating the action of hLigI during Okazaki fragment processing and ligation and the essential role of PCNA in efficient fork progression (17,40). At the same time, replication was slightly hindered by the presence of CTG/CAG repeats in the template, an effect further exacerbated by hLigI-altered backgrounds.…”

“…This increase was dependent upon the presence of a functional PCNA interaction motif but not linked to ligation activity (31). (ii) An excess of hLigI, either full-length or its aminoterminal region (required for PCNA interaction) but not the catalytic domain, inhibited in vitro SV40 DNA replication by HeLa cell extracts (40). This disruption by the non-catalytic domain of hLigI was interpreted to be because of an imbalanced availability of hLigI-interacting partners, like PCNA.…”

“…1) (18). Because it has been suggested that the steady state levels of hLigI protein may affect replication or repair pathways (40) and hLigI is frequently overexpressed in human malignant processes (48), we also examined its levels in the three 46BRLigI cell lines (18). Interestingly, different levels of hLigI were observed ( Fig.…”

Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III

“…LigI is the major replicative ligase with limited functions in some DNA repair pathways (1)(2)(3)(4). LigIV joins DNA double-stranded breaks in the nonhomologous end joining and during V(D)J recombination in the developing immune system (5).…”