Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol ␦ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol ␦. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol ␦ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol ␦, disrupts the DNA ligase I-PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA-DNA pol ␦ complex, DNA pol ␦ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.DNA-joining events are required during the replication of the mammalian genome because of the discontinuous nature of lagging strand DNA synthesis. There is substantial biochemical evidence indicating that DNA ligase I, which is one of four DNA ligases in mammalian cells (1, 2), is the enzyme responsible for joining Okazaki fragments. The involvement of DNA ligase I in DNA replication was first suggested by studies demonstrating an elevated level of this enzyme activity in regenerating liver compared with normal liver (3). More recently, DNA ligase I has been identified as a component of a high molecular weight replication complex (4, 5) and has been shown to efficiently join Okazaki fragments in a DNA replication assay with other highly purified replication proteins (6, 7).In agreement with this putative role in DNA replication, human DNA ligase I cDNA complements the conditional lethal phenotype of a Saccharomyces cerevisiae cdc9 DNA ligase mutant (8), and LIG1 homozygous null mouse embryonic stem cells are viable only when a full-length DNA ligase I cDNA is ectopically expressed (9). Moreover, a DNA ligase I mutant human cell line, 46BR, exhibits abnormal joining of Okazaki fragments (10-13). The replication defect in extracts from this cell line can be complemented by the addition of DNA ligase I but not by the addition of DNA ligase III or T4 DNA ligase (14).DNA polymerases ␣ and ␦, replication protein A, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), RNase H, FEN1, and DNA...

In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of~20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21 CIP1/WAF1 , we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.

An apurinic/apyrimidinic (AP) site is one of the most abundant lesions spontaneously generated in living cells and is also a reaction intermediate in base excision repair. In higher eukaryotes, there are two alternative pathways for base excision repair: a DNA polymerase ␤-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Here we have reconstituted PCNA-dependent repair of AP sites with six purified human proteins: AP endonuclease, replication factor C, PCNA, flap endonuclease 1 (FEN1), DNA polymerase ␦, and DNA ligase I. The length of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides, although longer patches of up to seven nucleotides could be detected. Neither replication protein A nor Ku70/80 enhanced the repair activity in this system. Disruption of the PCNAbinding site of either FEN1 or DNA ligase I significantly reduced efficiency of AP site repair but did not affect repair patch size.Base excision repair is the major mechanism that replaces bases having relatively small modifications, e.g. deoxyuracil, thymine glycol, or 8-oxo-guanine. In this process, the damaged bases are recognized and removed by specific DNA-N-glycosylases, producing apurinic/apyrimidinic (AP) 1 sites as common intermediates. AP sites are also generated by spontaneous depurination/depyrimidination and by the action of many different DNA damaging agents. AP sites are one of the most abundant DNA lesions in living cells. Base excision repair further processes AP sites to complete repair. The primary mechanism for AP site repair includes the following reactions: 1) incision of the AP site to generate a nick with 3Ј-OH and 5Ј-deoxyribose phosphate (dRP) termini; 2) DNA synthesis at the incised site; 3) excision of the dRP group from the 5Ј terminus; and 4) DNA ligation. Recent studies of in vitro repair systems derived from higher eukaryotes demonstrated that repair of AP sites could proceed by two alternative pathways: a DNA polymerase ␤ (pol ␤)-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway (1, 2).The pol ␤-dependent pathway requires three purified factors: AP endonuclease, pol ␤, and DNA ligase (3). To repair AP sites in this pathway, pol ␤ catalyzes two reactions: DNA synthesis by a DNA polymerase activity and excision of the 5Ј-dRP group by a dRP lyase activity (4). Thus, the pol ␤-dependent pathway can efficiently repair unmodified natural AP sites, but cannot repair modified AP sites which are refractory to ␤-elimination catalyzed by pol ␤/dRP lyase. To permit repair of the modified AP sites, the flap endonuclease 1 (FEN1) removes the dRP group and its 3Ј-adjacent nucleotide(s) by hydrolysis (5, 6). Finally, DNA ligase (either ligase I or the ligase III␣-XRCC1 complex in higher eukaryotes) seals the phosphodiester backbone to complete the repair reaction (3,7,8). Involvement of these enzymes in base excision repair in vivo is supported by studies with mutant mammalian cell lines. Pol ␤-deficient cells are hypersensit...

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, ␣ and ␤, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-␣ cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-␤ cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-␤ mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. The joining of DNA single-strand breaks is an essential step in the completion of lagging-strand DNA synthesis and DNA excision repair pathways. Additionally, exchanges between homologous DNA duplexes, which are completed by the cleavage of Holliday junctions, require DNA joining events to generate intact recombinant molecules.Three human genes encoding DNA ligases have been identified (4, 10, 45). Genetic and biochemical studies on the product of the LIG1 gene indicate that this enzyme functions to join Okazaki fragments during DNA replication (4,5,30,36,42,46). The sensitivity of the DNA ligase I-mutant cell line 46BR to DNA damage by alkylating agents and the abnormal repair of uracil-containing DNA substrates by 46BR cell extracts implicate DNA ligase I in DNA base excision repair (5,20,24,30,38). The recent characterization of an interaction between DNA polymerase ␤, which is essential for base excision repair of alkylation damage in mammalian cells (35), and DNA ligase I within a multiprotein complex that catalyzes the repair of a uracil-containing DNA substrate provides evidence at the molecular level that DNA ligase I is involved in DNA base excision repair (29).The LIG3 and LIG4 genes encode polypeptides that have similar electrophoretic mobilities in denaturing polyacrylamide gels (45). These gene products, with molecular masses of about 100 kDa, can be distinguished by the ability of DNA ligase III to form a stable complex with the product of the human XRCC1 gene (8,9,45). Human XRCC1 was cloned by its ability to complement the hypersensitivity of the Chinese hamster ovary cell line EM9 to DNA-alkylating agents (39, 40). Because the EM9 cell line is defective in the joining of DNA single-strand breaks and contains reduced levels of DNA ligase III activity, it appears that DNA ligase III functions in the repair of DNA single-strand breaks that arise either by the direct action of a DNA-damaging agent, such as ionizing radiation, or as a consequence of DNA repair enzymes excising lesions (8,9,25,39,40). At present, very little is known about the cellular role of DNA ligase IV.Analysis of the steady-state levels of DNA ligases I and III mRNAs in different human tissues and cells revealed that both of these genes are highly expressed in the testis. In ...