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The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2
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The MOI5 gene encodes the catalytic subunit of a protein kinase that zc.*.,.'va~escdc2 and other cyclindependent kinases (CDKs) through phosphorylation of Thr161 and its homologues D. FESQUET ETAL.
EMBOJ. 12, 3111-3121
The cdc2-related protein p40MO15is the catalytic subunit of a protein kinase that can activate p33cdk2and p34 cdc2 R.Y.C. POON ETAL.
EMBOJ. 12, 3123-3132
CAK, the p34cdczactivating kinase, contains a protein identical or closely related to p40 Mo15 M.J. SOLOMON, J.W. HARPER AND J. SHUTTLEWORTH
EMBOJ. 12, 3133-3142
The control of the cell division cycle in eukaryotic cells involves oscillations in the activity of protein kinases consisting of complexes between cdc2-1ike catalytic subunits and cyclins. The kinase activity of these complexes is cyclin-dependent and regulated by phosphorylation. In the cdc2 paradigm, phosphorylation of Thrl4 or Tyrl5 inhibits activity, whereas phosphoryla¢ion of Thrl61 is required for kinase activity. Thrl61 phosphorylation is c-~talysed by a kinase known as cdc2/cdk2 activating kinase (CAK). These papers demon-
Greater susceptibility to mutations in lagging strand of DNA replication in Escherichia coil than in leading strand X. VEAUTE AND R.P.P. FUCHS
Science 261,598-600
The way mutagenic lesions are handled in leading- and lagging-strand replication may have implications not only tor mutagenesis but also for understanding the fine detail of the mechanistic dif-
Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells T. FEATHERSTONE AND C. HUXLEY
Genomics 17, 267-278
There have been many reports of transfer of yeast artificial chromosomes (YACs) to mammalian cells, in which the YACs integrated as stable components of the host cell chromosomes. Featherstone and Huxley have found that, in some c,:ll lines generated by fusing mouse cells with YAC-containing
strate that MO15, the product of a cdc2-related kinase gene previously cloned from a Xenopus cDNA library, is the catalytic subunit of CAK. The initial approach of all three groups was to purify the activating kinase from starfish, human or frog extracts, respectively, using standard fractionation techniques and assays that could detect activation of p33cdk2 or p34 cdc2 by phosphorylation of Thrl61. Fesquet et al. purified CAK from starfish and found a complex containing two polypeptides: p37 and p40. From partial peptide sequence p37 was identified as the starfish homologue of frog p40 MO15. In a different approach, Poon et aL found that the MO15 protein kinase specifically phosphorylated proteins from germinal vesicle extracts, where it is localized, with M, in the range 32-34 kDa, suggesting that the cdc2/cdk2 family could be targets of the MO15 protein kinase. Solomon et al. used immunoblotting to demonstrate that MO15 was present in their most purified fractions, and that its presence was consistent with CAK activity. Once the identification of MO15 had been made, all three groups used an antibody specific to MO15 to confirm that MO15 was a component of CAK. Immunoprecipitates containing MO15 catalyse phosphate transfer from ATP, or GTP, to Thrl61 (or equivalent Thr residue) of cdc2 and cdk2, thereby activating them. In addition, Pooh et
aL showed that recombinant MOI5 produced in bacteria could also activate cdk2, but could only do so if first activated itself by exposure to frog extract in the presence of ATP. Thus, CAK is itself regulated by phosphorylation and/or complex formation. Activation of cdc2/cdk2 is also cyclindependent, but not cyclin-specific, since cyclins A, B or E were sufficient for activation. Fesquet et aL found that the presence of cyclin was necessary for phosphorylation of Thrl61 by CAK, suggesting that the kinasecyclin complex was the target of CAK. However, Poon et al. found that cyclin was not necessary for phosphorylation of Thrl61. This probably reflects differences in the sources of reagents used, some of which may contain other regulatory factors. The conclusion that MO15 is the catalytic subunit of CAK was surprising, first because MO15 was previously shown to delay cdc2 activation during meiosis, an inhibitory rather than activating role. Second, since MO15 is itself a close member of the cdc2/cdk2 protein kinase family, its behaviour seems rather incestuous. The other nearest equivalents of MO15 are the rice cdc2 homologue and the KIN28 protein of budding yeast. These three proteins vary slightly from cdc2/cdk2 in the residues around Thrl61 or its equivalent. Thus they may represent the catalytic subunits of CAK-like rather than cdc2/cdk2-1ike kinases. ,~
ferences between leading- and laggingstrand replication. Veaute and Fuchs have introduced a mutagenic lesion lbinding of the chemical carcinogen N-2-acetylaminofluorene (AAF) to the C-8 of guaninel into two different mutagenic 'hot spot' positions on the nontranscribed strand of a gene carried on the pUC8 plasmid of E. coil Mutagenesis induced by the lesion was measured with the gene either in its normal orientation, or reversed so that the leading
strand became the lagging strand and vice versa. The mutation frequency (single- or two-nucleotide deletions, both of which cause frame shifts) was around 20-fold higher when the adduct was on the lagging strand. The results may indicate that the polymerase complex can elongate more rapidly from a 'slipped mutagenic intermediate' (SMI) on the lagging strand, thereby stabilizing the SMI over reversion to the correct primer-template alignment.
yeast spheroplasts, both YACs and yeast genomic DNA appeared as extrachromosomal elements in the fusion cell lines. Retention of the YACs for a limited number of cell divisions indicated that they had some replicative ability but segregated poorly. Manipulation of these YACs might help to reveal the elements needed for replication and segregation of DNA in mammalian cells. In their appearance and behaviour, the extrachromosomal YACs behaved like double-minutes - centromere-less
circular elements that contain amplified DNA. In a few cell lines the YACs integrated into the mouse chromosomes, where they formed large blocks similar to so-called 'homogeneously stained regions' (HSRs), which are also characteristic of amplified chromatin. Double minutes and HSRs are frequently observed in cancer ceils or after prolonged selection for drug resistance; it is interesting that they can apparently be formed when DNA is introduced into cells by cell fusion, a~
TIG OCTOBER1993 VOL.9 NO. 10
m
The MOI5 gene encodes the catalytic subunit of a protein kinase that zc.*.,.'va~escdc2 and other cyclindependent kinases (CDKs) through phosphorylation of Thr161 and its homologues D. FESQUET ETAL.
EMBOJ. 12, 3111-3121
The cdc2-related protein p40MO15is the catalytic subunit of a protein kinase that can activate p33cdk2and p34 cdc2 R.Y.C. POON ETAL.
EMBOJ. 12, 3123-3132
CAK, the p34cdczactivating kinase, contains a protein identical or closely related to p40 Mo15 M.J. SOLOMON, J.W. HARPER AND J. SHUTTLEWORTH
EMBOJ. 12, 3133-3142
The control of the cell division cycle in eukaryotic cells involves oscillations in the activity of protein kinases consisting of complexes between cdc2-1ike catalytic subunits and cyclins. The kinase activity of these complexes is cyclin-dependent and regulated by phosphorylation. In the cdc2 paradigm, phosphorylation of Thrl4 or Tyrl5 inhibits activity, whereas phosphoryla¢ion of Thrl61 is required for kinase activity. Thrl61 phosphorylation is c-~talysed by a kinase known as cdc2/cdk2 activating kinase (CAK). These papers demon-
Greater susceptibility to mutations in lagging strand of DNA replication in Escherichia coil than in leading strand X. VEAUTE AND R.P.P. FUCHS
Science 261,598-600
The way mutagenic lesions are handled in leading- and lagging-strand replication may have implications not only tor mutagenesis but also for understanding the fine detail of the mechanistic dif-
Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells T. FEATHERSTONE AND C. HUXLEY
Genomics 17, 267-278
There have been many reports of transfer of yeast artificial chromosomes (YACs) to mammalian cells, in which the YACs integrated as stable components of the host cell chromosomes. Featherstone and Huxley have found that, in some c,:ll lines generated by fusing mouse cells with YAC-containing
strate that MO15, the product of a cdc2-related kinase gene previously cloned from a Xenopus cDNA library, is the catalytic subunit of CAK. The initial approach of all three groups was to purify the activating kinase from starfish, human or frog extracts, respectively, using standard fractionation techniques and assays that could detect activation of p33cdk2 or p34 cdc2 by phosphorylation of Thrl61. Fesquet et al. purified CAK from starfish and found a complex containing two polypeptides: p37 and p40. From partial peptide sequence p37 was identified as the starfish homologue of frog p40 MO15. In a different approach, Poon et aL found that the MO15 protein kinase specifically phosphorylated proteins from germinal vesicle extracts, where it is localized, with M, in the range 32-34 kDa, suggesting that the cdc2/cdk2 family could be targets of the MO15 protein kinase. Solomon et al. used immunoblotting to demonstrate that MO15 was present in their most purified fractions, and that its presence was consistent with CAK activity. Once the identification of MO15 had been made, all three groups used an antibody specific to MO15 to confirm that MO15 was a component of CAK. Immunoprecipitates containing MO15 catalyse phosphate transfer from ATP, or GTP, to Thrl61 (or equivalent Thr residue) of cdc2 and cdk2, thereby activating them. In addition, Pooh et
aL showed that recombinant MOI5 produced in bacteria could also activate cdk2, but could only do so if first activated itself by exposure to frog extract in the presence of ATP. Thus, CAK is itself regulated by phosphorylation and/or complex formation. Activation of cdc2/cdk2 is also cyclindependent, but not cyclin-specific, since cyclins A, B or E were sufficient for activation. Fesquet et aL found that the presence of cyclin was necessary for phosphorylation of Thrl61 by CAK, suggesting that the kinasecyclin complex was the target of CAK. However, Poon et al. found that cyclin was not necessary for phosphorylation of Thrl61. This probably reflects differences in the sources of reagents used, some of which may contain other regulatory factors. The conclusion that MO15 is the catalytic subunit of CAK was surprising, first because MO15 was previously shown to delay cdc2 activation during meiosis, an inhibitory rather than activating role. Second, since MO15 is itself a close member of the cdc2/cdk2 protein kinase family, its behaviour seems rather incestuous. The other nearest equivalents of MO15 are the rice cdc2 homologue and the KIN28 protein of budding yeast. These three proteins vary slightly from cdc2/cdk2 in the residues around Thrl61 or its equivalent. Thus they may represent the catalytic subunits of CAK-like rather than cdc2/cdk2-1ike kinases. ,~
ferences between leading- and laggingstrand replication. Veaute and Fuchs have introduced a mutagenic lesion lbinding of the chemical carcinogen N-2-acetylaminofluorene (AAF) to the C-8 of guaninel into two different mutagenic 'hot spot' positions on the nontranscribed strand of a gene carried on the pUC8 plasmid of E. coil Mutagenesis induced by the lesion was measured with the gene either in its normal orientation, or reversed so that the leading
strand became the lagging strand and vice versa. The mutation frequency (single- or two-nucleotide deletions, both of which cause frame shifts) was around 20-fold higher when the adduct was on the lagging strand. The results may indicate that the polymerase complex can elongate more rapidly from a 'slipped mutagenic intermediate' (SMI) on the lagging strand, thereby stabilizing the SMI over reversion to the correct primer-template alignment.
yeast spheroplasts, both YACs and yeast genomic DNA appeared as extrachromosomal elements in the fusion cell lines. Retention of the YACs for a limited number of cell divisions indicated that they had some replicative ability but segregated poorly. Manipulation of these YACs might help to reveal the elements needed for replication and segregation of DNA in mammalian cells. In their appearance and behaviour, the extrachromosomal YACs behaved like double-minutes - centromere-less
circular elements that contain amplified DNA. In a few cell lines the YACs integrated into the mouse chromosomes, where they formed large blocks similar to so-called 'homogeneously stained regions' (HSRs), which are also characteristic of amplified chromatin. Double minutes and HSRs are frequently observed in cancer ceils or after prolonged selection for drug resistance; it is interesting that they can apparently be formed when DNA is introduced into cells by cell fusion, a~
TIG OCTOBER1993 VOL.9 NO. 10
m