Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their Nand C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis. INTRODUCTIONMany aspects of eukaryotic cell cycle progression depend on the maintenance and modulation of chromosome architecture. The structural maintenance of chromosomes (SMC) superfamily comprises proteins with important functions in a number of chromosome maintenance activities, including chromosome condensation, sister chromatid cohesion, and DNA repair (reviewed by Hirano, 1998;Hirano, 1999;Strunnikov and Jessberger, 1999;Cobbe and Heck, 2000;Nasmyth et al., 2000). In eukaryotes, six classes of SMC protein have been identified. These are the four classical SMC subfamilies (SMC1-SMC4), named for their Saccharomyces cerevisiae members, along with the somewhat more distantly related Schizosaccharomyces pombe Rad18 (SMC6) and Spr18 (SMC5) groups (Jessberger et al., 1998;Fousteri and Lehmann, 2000).All these SMC proteins share the same characteristic structure, in which two extended coiled-coil domains, separated by a short hinge region, are flanked by highly conserved globular head and tail domains ( Figure 1A). The ␣-helical coiled-coils are involved in protein-protein interactions and the N-and C-terminal domains are thought to mediate ATP binding and hydrolysis (Jessberger et al., 1998). Members of the SMC family form heterodimers, which probably adopt an antiparallel conformation (Melby et al., 1998).SMC heterodimers associate with a number of additional proteins to form high molecular mass functional complexes. In budding yeast, the Smc1/Smc3 heterodimer forms the core of the cohesin complex, which is required to hold replicated sister chromatids together until their segregation at anaphase (Guacci et al., 1997;Michaelis et al., 1997). An equivalent cohesin complex has been identified in Xenopus laevis egg extracts (Losada et al., 1998) and human cells (Schmiesing et al., 1998). In bovine cel...

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