A Physical and Functional Interaction between Yeast Pol4 and Dnl4-Lif1 Links DNA Synthesis and Ligation in Nonhomologous End Joining

Abstract

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...

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“…2 The present study suggests that Pol , with its unusually high rate for misalignment-mediated errors and its preference for short gap substrates, also appears to be well suited to participate in NHEJ. The possible involvement of Pol in this process is further suggested by genetic (50) and biochemical studies (51) indicating that in yeast, end-joining depends upon the function of DNA Pol IV, a close homologue of human Pol . In a recent study, Heidenreich et al (52) showed that NHEJ contributes to mutagenesis in non-replicating diploid yeast cells.…”

“…This specificity is characteristic of Pol and thus suggests the involvement of Pol IV. The end-joining activity of Pol IV requires the presence of its N-terminal BRCT domain (50,51). It has been shown that through the BRCT domain, Pol IV interacts with Dnl4, a subunit of the Dnl4-Lif1 complex (51), which is the homologue of the human ligase IV-XRCC4 complex.…”

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“…A third domain, the breast cancer carboxy-terminal (BRCT)-associated domain is also found in many family X members, including the single Pol X members in S. cerevisiae [Tseng and Tomkinson, 2002] and Schizosaccharomyces pombe [Gonzalez-Barrera et al, 2005] and three out of the four Pol X members (not Pol b) in vertebrates [Aoufouchi et al, 2000]. Pol X members have a single BRCT domain, unlike many BRCT domain-containing proteins (including ligase IV) where the domains are present as one or more tandemly repeated pairs [Leung and Glover, 2011].…”

“…As with other BRCT domains, the BRCT domain in TdT can also interact with a phosphoserine-containing motif, but whether this has functional relevance is not yet clear [Yu et al, 2003]. Rather, in all examples tested (Pol4 [Tseng and Tomkinson, 2002] as well as vertebrate Pol k [Fan and Wu, 2004;Lee et al, 2004;Ma et al, 2004;Nick McElhinny et al, 2005;Mueller et al, 2008], Pol l [Mahajan et al, 2002;Ma et al, 2004;Nick McElhinny et al, 2005;DeRose et al, 2007] and TdT), the BRCT domain is required for physical interaction with Ku and XRCC4-ligase IV at DNA ends, and there is as yet Environmental and Molecular Mutagenesis. DOI 10.1002/em no contribution of Ku, XRCC4, or ligase IV phosphorylation.…”

“…Both yeast orthologs are primarily template dependent, yet are unusually able to accommodate mismatches near primer termini, consistent with the need for NHEJ Pols to extend from minimally aligned primer/template substrates. S. cerevisiae Pol4 physically associates with the NHEJ ligase (Dnl4) through its BRCT domain, and it can fill in gaps generated by alignment of complementary ends before ligation [Tseng and Tomkinson, 2002]. …”