The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.

Abstract

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia-and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are-300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination. [ Mitotic cells guard against genetic instability and increase their survival following DNA damage both by direct DNA repair mechanisms and by delaying progression through the cell cycle. Depending on the position of the cell within the cell cycle at the time of irradiation, DNA damage in mammalian cells can effect specific checkpoints that prevent passage from G 1 to S phase, progression through S phase, or passage from G 2 into mitosis. These checkpoints are thought to prevent dele-terious events such as replication of damaged DNA or the segregation of fragmented chromosomes during mi-tosis (Hartwell and Kastan 1994) and therefore assure the accurate transmission of the genetic material.

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

“…DNA-PK is clearly a conventional protein kinase and catalyses the phosphorylation of a number of proteins (Hoekstra, 1997). In the case of ATR, several workers have reported autophosphorylation of the 4250 kDa polypeptide (Cliby et al, 1998;Keegan et al, 1996) in immunoprecipitates, which was dependent on a functional catalytic domain (Cliby et al, 1998). However this is not proof that ATR is a conventional protein kinase, as both classical PI-3 kinase (Carpenter et al, 1993;Dhand et al, 1994) and the yeast VPS34 lipid kinase (Stack and Emr, 1994) are capable of carrying out speci®c autophosphorylation, but are clearly lipid kinases in vivo.…”

“…This protein, termed ATR (for ATM and rad3 related) can complement the UV sensitivity of mec1 mutants when expressed in S. cerevisiae, and expression of kinase-dead ATR protein in human cells causes sensitivity to DNA damaging agents and defects in cell cycle checkpoints (Cliby et al, 1998;Wright et al, 1998). Immunoprecipitates of ATR have associated protein kinase activity which is reduced in immunoprecipitates of putative kinase-dead protein (Canman et al, 1998;Keegan et al, 1996;Sarkaria et al, 1998;Tishko et al, 1997).…”

Sex chromosomes, synapsis, and cohesins: a complex affair

“…The list includes: XY40 (Smith and Benavente 1992;Alsheimer et al 1997), BRCA1 (Scully et al 1997), ATR (Keegan et al 1996), XMR (Escalier and Garchon 2000), and also Xist RNA, a nontranslated RNA involved in the inactivation of the X chromosomes in female somatic cells and X and Y chromosomes in male meiotic cells (Ayoub et al 1997). While the function of many of these proteins remains uncertain, recent works have yielded important clues as regards the roles of BRCA1 and ATR.…”

Rhabdomyosarcoma – working out the pathways

“…ATR is the mammalian homolog of the yeast rad3 gene, and is related to the human ataxia telangiectasia mutated (ATM) gene; both are nuclear factors with phosphatidylinositol 3-kinase-related activity (Keegan et al, 1996). When overexpressed in myoblasts ATR inhibits MyoD-dependent di erentiation, and induces aberrant centrosome ampli®cation, aneuploidy, and loss of girradiation-induced G 1 arrest.…”