A newly identified DNA ligase of <i>Saccharomyces cerevisiae</i>involved in <i>RAD52</i>-independent repair of DNA double-strand breaks

The repair of DNA double-strand breaks is critical for maintaining genetic stability. In the non-homologous end-joining pathway, DNA ends are brought together by end-bridging factors. However, most in vivo DNA double-strand breaks have terminal structures that cannot be directly ligated. Thus, the DNA ends are aligned using short regions of sequence microhomology followed by processing of the aligned DNA ends by DNA polymerases and nucleases to generate ligatable termini. Genetic studies in Saccharomyces cerevisiae have implicated the DNA polymerase Pol4 and the DNA structurespecific endonuclease FEN-1(Rad27) in the processing of DNA ends to be joined by Dnl4/Lif1. In this study, we demonstrated that FEN-1(Rad27) physically and functionally interacted with both Pol4 and Dnl4/Lif1 and that together these proteins coordinately processed and joined DNA molecules with incompatible 5 ends. Because Pol4 also interacts with Dnl4/Lif1, our results have revealed a series of pair-wise interactions among the factors that complete the repair of DNA doublestrand breaks by non-homologous end-joining and provide a conceptual framework for delineating the endprocessing reactions in higher eukaryotes.The repair of DNA double-strand breaks (DSBs) 1 is critical for the maintenance of genomic integrity and stability. There are two main DSB repair pathways in eukaryotes: homologous recombination and non-homologous end joining (NHEJ) (1). In the error-free homologous recombination pathway, an intact homologous DNA duplex acts as a template for repair, resulting in the accurate restoration of the broken DNA molecule. By contrast, in NHEJ, the two broken ends are simply rejoined to one another in a process that frequently causes loss and/or gain of nucleotides at the break site and occasionally results in the joining of previously unlinked DNA molecules. Notably, defects in the repair of DSBs by either homologous recombination or NHEJ have been linked with cancer predisposition (2, 3).Studies with mammalian cells identified the DNA-dependent protein kinase (DNA-PK), which is composed of the DNA end binding Ku70/Ku80 heterodimer and the DNA-PK catalytic subunit, and the DNA ligase IV/XRCC4 complex as key NHEJ factors (4). In Saccharomyces cerevisiae, Hdf1/Hdf2 and Dnl4/Lif1 are the functional homologs of Ku70/Ku80 and DNA ligase IV/XRCC4, respectively (4). Although yeast lacks a DNA-PK catalytic subunit homolog, genetic studies have revealed that the Rad50/Mre11/Xrs2 complex is a key player in NHEJ (4 -7). Using purified protein complexes, it has been shown that the Rad50/Mre11/Xrs2 complex has robust endbridging activity and specifically stimulates intermolecular DNA joining by Dnl4/Lif1 (8). Furthermore, efficient intermolecular DNA joining mediated by the Rad50/Mre11/Xrs2 and Dnl4/Lif1 complexes was dependent upon Hdf1/Hdf2 at physiological salt concentrations (8). Based on these results, it seems likely that Hdf1/Hdf2 enhances the recruitment of the Rad50/ Mre11/Xrs2 end-bridging complex to DNA ends and that Dnl4/ Lif1 joins the res...

Section: Discussionmentioning

confidence: 99%

Section: Coordinated Gap-filling Synthesis and Flap Cleavage Bymentioning

confidence: 99%

Processing and Joining of DNA Ends Coordinated by Interactions among Dnl4/Lif1, Pol4, and FEN-1

Tseng

Tomkinson

2004

“…The physiologic phosphorylation target of the 469-kDa DNA-PK cs has not been identified (10). After the DNA ends are trimmed into a form that is ligatable, a complex of XRCC4 and DNA ligase IV is responsible for carrying out the ligation specifically of double-strand breaks (11)(12)(13)(14)(15)(16)(17).…”

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confidence: 99%

Binding of Inositol Hexakisphosphate (IP6) to Ku but Not to DNA-PKcs

Lieber

2002

The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK cs , to that end. The Ku⅐DNA-PK cs complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP 6 ) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP 6 bound to DNA-PK cs (not to Ku). Here we clearly show that, in fact, IP 6 associates not with DNA-PK cs , but rather with Ku. Furthermore, the binding of DNA ends and IP 6 to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

“…In the lower eukaryote Saccharomyces cerevisiae, the DNA ligases encoded by the CDC9 and DNL4 genes are functional homologs of the LIG1 and LIG4 genes. However, this organism apparently lacks a LIG3 gene homolog (2)(3)(4)(5).…”

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confidence: 99%

Interactions of the DNA Ligase IV-XRCC4 Complex with DNA Ends and the DNA-dependent Protein Kinase

Chen

Trujillo

Sung

et al. 2000

204

145

Three genes encoding DNA ligases, LIG1, LIG3, and LIG4, have been identified in the mammalian genome (1). The products of these genes appear to be responsible for the DNAjoining events that complete DNA replication, genetic recombination, and DNA repair in mammalian cells. In the lower eukaryote Saccharomyces cerevisiae, the DNA ligases encoded by the CDC9 and DNL4 genes are functional homologs of the LIG1 and LIG4 genes. However, this organism apparently lacks a LIG3 gene homolog (2-5).Mammalian DNA ligases consist of a conserved catalytic domain flanked by unrelated sequences (1). It has been suggested that protein-protein interactions mediated by the unique regions flanking the catalytic domain determine the cellular functions of these enzymes. In support of this hypothesis, recent studies have identified specific protein partners for each of the LIG gene products. The non-catalytic N-terminal domain of DNA ligase I interacts with both proliferating cell nuclear antigen and DNA polymerase ␤, implicating this species of DNA ligase in DNA replication and excision repair pathways (6, 7). In contrast, the non-catalytic C-terminal extensions of DNA ligases III␣ and IV contain BRCT motifs (8), a putative protein-protein interaction domain first identified in the protein encoded by the breast cancer susceptibility gene BRCA1 (9, 10). The single BRCT motif of DNA ligase III␣ mediates complex formation with the DNA repair protein XRCC1, linking this species of DNA ligase with base excision repair and the repair of DNA single-strand breaks (11,12). Similarly, the region of DNA ligase IV containing tandem BRCT motifs is involved in complex formation with the DNA repair protein XRCC4,implicating DNA ligase IV in non-homologous end joining (NHEJ) 1 and V(D)J recombination (13, 14). Human individuals with mutations in either the LIG1 (15) or LIG4 gene (16) have been identified. The initial symptoms of the individual with DNA ligase I deficiency were recurrent infections caused by severe combined immunodeficiency (17). Cell lines established from this patient exhibited defects in Okazaki fragment joining and sensitivity to a wide range of DNA-damaging agents, a phenotype consistent with the predicted role of DNA ligase I in DNA replication and excision repair (18,19). When the DNA ligase I-deficient cells were activated for V(D)J recombination by ectopic expression of the RAG proteins, there was no apparent defect in this type of recombination (20,21). However, studies with cell-free extracts suggest that DNA ligase I contributes to the completion of V(D)J recombination (22).The individual with a mutated LIG4 gene presented with leukemia. Unfortunately, attempts to treat this disease with chemotherapy and then ionizing radiation caused a severe reaction (16,23). The extreme radiosensitivity of cell lines established from this individual is consistent with the predicted role of DNA ligase IV in the repair of DNA double-strand breaks by NHEJ. Surprisingly, the DNA ligase IV-deficient individual did not appear to be immunodefi...