Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining. Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks. Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain. This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1. Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation. Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomologymediated pathway of nonhomologous end joining.Mechanisms for the repair of DNA double-strand breaks (DSBs) 1 can be divided into two classes based on the requirement for DNA sequence homology. In the major homologydependent pathway, repair involves an intact duplex that is homologous to the broken molecule. This is the major DSB repair pathway in the yeast Saccharomyces cerevisiae and is mediated by members of the RAD52 epistasis group that includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, XRS2, and RDH54/TID1 (1). Alternatively, broken DNA ends are simply brought together, processed, and then ligated by repair mechanisms, known collectively as nonhomologous end joining (NHEJ) (2). Unlike the major recombinational repair pathway that faithfully restores the genetic information, nonhomologous end joining frequently causes genetic alterations that range from the loss or addition of a few nucleotides at the break site to gross rearrangements such as chromosomal translocations (2).Genetic studies in S. cerevisiae have identified the products of the HDF1, HDF2, RAD50, MRE11, XRS2, DNL4, and LIF1 genes as key players in the major NHEJ pathway (3-14). HDF1 and HDF2 encode subunits of a heterodimeric DNA end-binding complex that is functionally homologous to the mammalian Ku70-Ku80 complex (3-6). Similarly, the Rad50-Mre11-Xrs2 and Dnl4-Lif1 complexes appear to be functional homologs of the hRad50-hMre11-NBS1 (7,8,(15)(16)(17)(18)(19)(20)(21) and DNA ligase IV-XRCC4 complexes (9 -13, 22, 23), respectively. Congruent with genetic analysis in yeast, a recent biochemical study has reconstituted DNA end joining with the purified NHEJ factors Hdf1-Hdf2, Rad50-Mre11-Xrs2, and Dnl4-Lif1 and demonstrated functional interactions among these complexes (24). Recently, a novel yeast NHEJ gene, NEJ1, has been identified, but the exact role of this gene product in NHEJ remains to be determined (25-28).Many of the gen...

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...

A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation.

A human HSMT3 cDNA encoding a homolog of the yeast SMT3, a suppressor of MIF2 mutations in a centromere protein gene, was identified and sequenced. The sequence of 95 amino acids deduced from the human HSMT3 cDNA exhibited 51.1% identity and 69.6% similarity to the yeast Smt3p sequence. The HSMT3 transcripts of 1.35Kb were found to be abundantly expressed in various human tissues.

Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [a- Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo-and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors.