Thirty percent of the 189 tumors studied to date express DNA polymerase ␤ variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein.Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorageindependent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase ␤ variant proteins, implying that it has a mutational basis. Because DNA polymerase ␤ functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.base excision repair ͉ DNA repair ͉ mutagenesis S pontaneous DNA damage occurs at a rate of Ϸ10,000 lesions per cell per day, and much of this damage is repaired by the base excision repair (BER) machinery (1, 2). The BER system plays a critical role in maintaining cellular genomic stability. During BER, damaged bases are removed by a DNA glycosylase, followed by incision of the DNA by AP endonuclease (APE) at a position that is usually 5Ј to the lesion, leaving a nick with a 3ЈOH and a 5Ј deoxyribose phosphate (2). DNA polymerase beta (pol ␤) binds to the nick, removes the deoxyribose phosphate with its DRP lyase activity (3), and fills in the single nucleotide gap, using its DNA polymerase activity (4).Fifty-eight of the 189 tumors characterized to date express DNA pol ␤ variant proteins (for review, see ref. 5) (6). Of these, 28 (48%) expressed variants with single amino acid alterations, seven expressed truncated forms of pol ␤, and eight expressed multiple variant forms of pol ␤. These mutations are absent from normal tissue from the same individuals and are not among the common polymorphisms found within the pol ␤ gene (http:͞͞ egp.gs.washington.edu͞data͞polb) (5, 7). In addition, an alternative splice variant of pol ␤ in which exon 11 is deleted was expressed in 15 tumors. This splice variant appears to interfere with BER (8). This exon 11 splice variant has been detected in normal tissue, including normal tissue isolated from 2 of 15 patients with tumors, and its link to cancer etiology remains controversial (9-12). Each of the tumors characterized to date also contain the wild-type (WT) pol ␤ allele.The I260M variant of pol ␤ was identified in a prostate carcinoma (13). Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymeras...

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant BudS and Ltel proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-s but inactive in promoting exchange on the Ras-related proteins Yptl and Rsrl. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP-or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.Ras proteins, like trimeric G proteins, are active when bound to GTP and inactive when bound to GDP (7). Activation of Ras proteins in vivo may be mediated by proteins referred to as guanine nucleotide exchange factors (also referred to as guanine nucleotide dissociation stimulators or releasing factors) (7). The first such exchange factor for Ras proteins to be identified was the CDC25 gene product of Saccharomyces cerevisiae (10,28 identified, BUD5 and LTEJ (11,26,35). Neither of these genes appears to function in the RAS pathway, but genetic analysis suggests that the BUD5 gene product acts as a GDP-GTP exchange factor for the yeast Rap homolog encoded by RSR1IBUD1 (1, 11). A much closer homolog of CDC25 exists in S. cerevisiae, the SDC25 gene (8). A C-terminal fragment of SDC25 expressed in Escherichia coli is a potent catalyst of GDP-GTP exchange for yeast Ras2 and mammalian p2lHras proteins, yet genetic analysis of SDC25 has not as yet revealed any cellular function for SDC25 in the control of Ras activity (13,14). Perhaps two distinct CDC25-like exchange factors in budding yeast cells allow Ras proteins to transduce signals from different growth stimuli.We undertook this study to determine more precisely which region of the lengthy CDC25 gene (1,589 codons) is required for essential cellular function and to use this information to test recombinant CDC25 protein fragments for GDP-GTP exchange activity. It has been suggested, on the basis of failure of prior attempts ...

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse‐specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG‐stimulated signaling pathway is important for synapse‐specific gene expression.

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