The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.

Genomic integrity is achieved by concerted functions of genes involved in all aspects of DNA metabolism throughout the cell cycle. Fundamental mechanisms for genome maintenance seem to be conserved throughout eukaryotes (16, 38, 39). However, given the large size and complexity of mammalian genomes, there are likely to be additional genes or pathways involved that remain to be discovered. Since mutations causing chromosome instability increase the risk of developing malignancies (14, 18, 45), exploring genes involved in genome maintenance is a first step to understand fundamental mechanisms of carcinogenesis. To uncover mammalian genes not previously known to have roles in maintaining genomic integrity, we have taken a forward genetics approach in mice. New chromosome instability mutations, arising from mouse N-ethyl-N-nitrosourea (ENU) mutagenesis (3), have been identified by a phenotype-based screen (37). In this screen, spontaneous micronucleus levels in erythrocytes, measured by a flow cytometric assay, were used to quantitate chromosome damage in vivo (11, 27). Micronuclei are derived from acentromeric chromosome fragments or from a whole chromosome having failed to be incorporated into the nuclei at mitosis, thereby representing chromosome breaks and aneuploidy (29). Spontaneous and radiation-induced chromosome instability in Atm (for ataxia telangiectasia mutated)-deficient mice can be detected as elevated levels of micronuclei in erythrocytes (37). To induce mutations that potentially cause chromosome instability, C57BL/6J males were mutagenized with the powerful germ line mutagen ENU (3, 13) and were bred in a classical three-generation scheme to produce descendants that are potentially homozygous for induced recessive mutations (37). These mice were subjected to a phenotype screen in which micronuclei in erythrocytes were semiautomatically measured by flow cytometry (6). Mutants were identified as outliers showing significantly higher numbers of spontaneous micronuclei. Using this screen, five independent mutations were identified among 763 pedigrees derived from the mutagenized males. chaos1 (for chromosome aberrations occurring spontaneously 1) was the first mutation identified in this screen. Treatment with radiation or mitomycin C (MMC) induced significantly higher frequencies of micronuclei in chaos1/chaos1 mice to a level that indicates hypersensitivity to agents inducing double-strand breaks (DSBs) or interstrand cross-links. This recessive mutation was genetically mapped on a 1.3 Mb interval on chromosome 16 (37). Among the genes residing in this region was Polq encoding DNA polymerase θ (theta). POLQ is homologous to Drosophila MUS308 that is believed to be involved in DNA interstrand cross-link repair (5, 10, 21). Orthologs apparently do not exist in single celled organisms such as bacteria and yeast. POLQ is also unique in that it contains helicase and polymerase domains near the N and C termini, respectively (10, 34, 37). There are at least 15 different DNA polymerases in higher eukaryotes (15, 36), and POLQ belongs to the “A” family. Whereas classical replicative DNA polymerases (α, δ, and ɛ) are essential for chromosomal DNA replication, recently discovered DNA polymerases are specialized for different cellular processes (15, 36). The most characteristic feature of these novel DNA polymerases (such as those of “Y” family) is the ability to bypass DNA lesions that block replicative DNA polymerases, termed translesion synthesis (7, 32, 33). There have been two biochemical studies on the DNA polymerase activity of human POLQ in vitro. Although one suggested a potentially unique translesion synthesis activity (22), it was not clear that the purified activity actually corresponded to full-length POLQ (34). Here, we conducted a genetic analysis of POLQ's involvement in maintenance of genomic integrity, which points to a potential role in recombinational repair. The Polq coding sequence in chaos1/choas1 mice was found to contain a de novo T-to-C transition, causing a serine-to-proline change (37). However, since this amino acid is not located in a conserved or critical region, it remained uncertain whether or not this mutation actually compromised protein function, to what extent, and if it was responsible for the micronucleus phenotype. Transgenic and knockout experiments reported here confirm the allelism of chaos1 to Polq. An enigmatic aspect of the chaos1 mice is the absence of overt phenotypes despite the elevated genomic instability in erythroblasts. To explore the suspected role in DSB repair, the chaos1 mutation was placed in a background deficient for ATM, which plays a key role in activating DNA damage signaling and cell cycle checkpoints (1), and in regulating DSB repair by homologous recombination (8, 28, 41-43). This resulted in synthetic semilethality, growth retardation, synergistic elevation in chromosome instability and, surprisingly, significantly delayed development of thymic lymphoma. The role(s) of POLQ in DSB repair and genome maintenance is discussed in light of these data.