Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in nucleotide biosynthesis and plays a central role in genome maintenance. Although a number of regulatory mechanisms govern RNR activity, the physiologic effect of RNR deregulation had not previously been examined in an animal model. We show here that overexpression of the small RNR subunit potently and selectively induces lung neoplasms in transgenic mice and is mutagenic in cultured cells. Combining RNR deregulation with defects in DNA mismatch repair, the cellular mutation correction system, synergistically increased RNR-induced mutagenesis and carcinogenesis. Moreover, the proto-oncogene K-ras was identified as a frequent mutational target in RNR-induced lung neoplasms. Together, these results show that RNR deregulation promotes lung carcinogenesis through a mutagenic mechanism and establish a new oncogenic activity for a key regulator of nucleotide metabolism. Importantly, RNR-induced lung neoplasms histopathologically resemble human papillary adenocarcinomas and arise stochastically via a mutagenic mechanism, making RNR transgenic mice a valuable model for lung cancer. [Cancer Res 2008;68(8):2652–60]

Steatoapoptosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and is an important factor in liver disease progression. We hypothesized that increased reactive oxygen species resulting ...

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1. Meiosis is a specialized cell division process in which germ cells undergo two cell divisions to produce haploid progeny. Two processes, genetic recombination and chromosome pairing/synapsis, are critical for successful meiosis and the production of gametes with high chromosomal integrity. The RAD9-RAD1-HUS1 (9-1-1) complex has been proposed to play critical roles in recombination as well as in the checkpoint-dependent monitoring of chromosomal synapsis by facilitating activation of the ATR checkpoint kinase. Our data indicate that HUS1 is required for normal germ cell development and fertility, for efficient completion of a subset of meiotic DNA recombination events, and for proper exclusion of the non-sex chromosomes from a specialized, repressive chromatin domain containing the X and Y chromosomes. However, HUS1 is not required for the meiotic functions of ATR in responding to chromosome synapsis defects. Furthermore, RAD1 localizes to sites along asynapsed chromosomes that lack detectable RAD9, and does so in the absence of Hus1, implicating RAD1 in a novel response to unsynapsed chromatin that is independent of the canonical 9-1-1 complex. Since mice lacking Hus1 in germ cells exhibit chromosomal abnormalities and severely reduced fertility, this work has broad implications for the maintenance of genome stability in the germline and for human reproductive health.

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.