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1.  Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. 
Nucleic Acids Research  1996;24(23):4639-4648.
Ku is a heterodimer of polypeptides of approximately 70 and 80 kDa (Ku70 and Ku80, respectively) that binds to DNA ends. Mammalian cells lacking Ku are defective in DNA double-strand break (DSB) repair and in site-specific V(D)J recombination. Here, we describe the identification and characterisation of YKU80, the gene for the Saccharomyces cerevisiae Ku80 homologue. Significantly, we find that YKU80 disruption enhances the radiosensitivity of rad52 mutant strains, suggesting that YKU80 functions in a DNA DSB repair pathway that does not rely on homologous recombination. Indeed, through using an in vivo plasmid rejoining assay, we find that YKU80 plays an essential role in illegitimate recombination events that result in the accurate repair of restriction enzyme generated DSBs. Interestingly, in the absence of YKU80function, residual repair operates through an error-prone pathway that results in recombination between short direct repeat elements. This resembles closely a predominant DSB repair pathway in vertebrates. Together, our data suggest that multiple, evolutionarily conserved mechanisms for DSB repair exist in eukaryotes. Furthermore, they imply that Ku binds to DSBs in vivo and promotes repair both by enhancing accurate DNA end joining and by suppressing alternative error-prone repair pathways. Finally, we report that yku80 mutant yeasts display dramatic telomeric shortening, suggesting that, in addition to recognising DNA damage, Ku also binds to naturally occurring chromosomal ends. These findings raise the possibility that Ku protects chromosomal termini from nucleolytic attack and functions as part of a telomeric length sensing system.
PMCID: PMC146307  PMID: 8972848
2.  Activation domains of transcription factors mediate replication dependent transcription from a minimal HIV-1 promoter. 
Nucleic Acids Research  1996;24(4):549-557.
Transcription from a minimal HIV-1 promoter containing the three Sp1 binding sites and TATA box can be activated without Tat by template DNA replication. Here we show that this activation can also be mediated by recombinant GAL4 fusion proteins containing the activation domains of Sp1, VP16 or CTF (or by full-length GAL4) targeted to the HIV-1 promoter by replacing the Sp1 sites with five GAL4 binding sites. Thus Sp1 is not unique in its ability to mediate replication activated transcription, although the degree of processivity elicited by the different activators varied significantly from strongly processive (GAL4-VP16) to relatively non-processive (GAL4-Sp1 or -CTF). Processive GAL4-VP16-activated transcription, but not efficient initiation, required multiple GAL4 binding sites. In the presence of Tat, transcription with GAL4-SP1 and GAL4-CTF was further activated (principally at the level of processivity) but GAL4-VP16-potentiated transcription was only slightly stimulated. The Tat-dependent switch from non-processive to fully processive transcription was particularly marked for GAL4-Sp1, an effect which may be relevant to the selection of Sp1 binding sites by the HIV-1 promoter.
PMCID: PMC145701  PMID: 8604293
3.  Lymphocyte subset infiltration patterns and HLA antigen status in colorectal carcinomas and adenomas. 
Gut  1996;38(1):85-89.
Fifty eight large bowel adenocarcinomas and 20 adenomas were studied immunohistochemically, using fresh frozen tissue sections, with regard to lymphocyte subpopulations (CD3, CD4, CD8, CD19, and CD20) in the inflammatory infiltrate and to expression of human leucocyte antigens (HLA-ABC, HLA-A2, and HLA-DR). The findings were related to differentiation and Duke's stage of carcinoma. The inflammatory infiltrate was found to have a phenotype that remained constant irrespective of the intensity of the inflammation. CD4 and CD3 positive cells predominated with fewer CD8 positive cells and a scanty diffuse CD19/20 positive cell population. CD19/20 follicular aggregates were common at the advancing margin of the carcinomas. There was no significant association with Duke's stage, differentiation or HLA status. HLA changes (ABC loss, A2 loss, and DR gain) were associated with differentiation, being more common and more extensive in poorly differentiated carcinomas. HLA-A2 loss was also associated with stage of progression of carcinoma. Inflammation associated with adenomas was found to have a similar phenotype to that associated with carcinomas. HLA changes in adenomas were uncommon, being seen in only one of our 20 cases.
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PMCID: PMC1382984  PMID: 8566865

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