Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C3 T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val 617 to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV. Polycythemia vera (PV)1 is a clonal myeloproliferative disorder characterized by increased production of red cells, granulocytes, and platelets (1-3). It mainly affects people between 40 and 60 years of age with an annual incidence of about 14 per million in the population. Thus far there is no effective cure for the disease. Phlebotomy is the mainstay of treatment for the disease, and hydroxyurea, interferon-␣, and anagrelide drug therapies and 32 P radiation therapy have commonly been used (3). The mortality rate is high if the disease is untreated or is associated with leukemia. Despite extensive studies in recent years, the molecular etiology of PV remains unknown.A major feature of PV is that hematopoietic progenitors in patients display hypersensitive responses to many growth factors and cytokines (1-3). Despite these abnormal responses, the numbers of receptors for the growth factors and cytokines on the surface of these cells are normal, suggesting a primary defect in a shared signaling pathway in these cells. Tyrosine phosphorylation is a fundamental regulatory mechanism for cell growth and development, and this process is controlled by coordinate actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs) (4). Both families of enzymes have highly diverse structures and functions. Their activities are tightly regulated under normal conditions, and deregulation of the enzymes produces human diseases. According to the human genomic data base, there are about 90 PTKs and 38 phosphotyrosine-specific PTPs (5-7). Both mutation of PTKs and PTPs have been implicated in human cancers. Recent studies using a large scale sequencing-based approach revealed that PTK and PTP genes are mutated more often in human cancers than previously anticipated. A minimum of 30% of colorectal cancers contains at least one mutation in PTKs, and 26% of them has a mutation in PTPs (8, 9). It is highly expected that mutations of PTKs and PTPs may also be a major manifestation of other types o...

The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.[Keywords: ATR; ATRIP; TopBP1; checkpoint; PIKK; Ddc2] Supplemental material is available at http://www.genesdev.org.

Graphical AbstractHighlights d HMCES senses abasic sites in ssDNA and forms a covalent DNA-protein crosslink d HMCES shields the abasic site from TLS polymerases and endonucleases d HMCES is a suicide enzyme and the DPC is ubiquitylated and degraded d HMCES is conserved in all domains of life and loss results in genetic instability In BriefAn enzyme conserved from bacteria to eukaryotes can covalently bind to abasic sites in single-stranded DNA to shield them and prevent genomic instability.

SUMMARY RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis, but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and͞or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.