The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. We now show that transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc(-/-) cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, we show that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. Our data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation.

Topoisomerase II␤-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase ⑀. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G 1 phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions. DNA polymerases (pol)1 play essential roles in chromosomal DNA replication and repair. In Saccharomyces cerevisiae three essential nuclear polymerases, ␣, ␦, and ⑀ have important functions in DNA replication. S. cerevisiae pol ⑀ is isolated as a complex of a catalytic subunit and three smaller subunits, Dpb2, 3, and 4 (1). This four-subunit structure is also conserved in the human enzyme, which consists of a catalytic subunit (2), a B subunit (3, 4), and two smaller subunits (5). Pol ⑀ is a proofreading DNA polymerase, which has been implicated in DNA replication, as temperature-sensitive mutants show defects in DNA replication in both S. cerevisiae and Schizosaccharomyces pombe (6 -8). Moreover, pol ⑀ is associated with origins of DNA replication and it proceeds along the replication fork (9). In human cells, pol ⑀ is associated with actively replicated cellular DNA (10) and has been shown to perform an important fraction of replicative DNA synthesis (11). Surprisingly, the catalytic domain of pol ⑀ is not essential for viability in S. cerevisiae. Instead, the C terminus, which interacts with Dpb2, exerts all of the essential functions (12).Pol ⑀ has been proposed to function in the repair of UVdamaged DNA because it is able to catalyze UV-induced DNA synthesis in vivo (13) and performs efficient gap-filling synthesis in the reconstituted nucleotide excision repair system (14). A role in base excision repair is suggested by the fact that pol ⑀ mutants fail to support repair synthesis in vitro, and repair activity can be restored by the addition of purified pol ⑀ (15). Pol ⑀ has also been proposed to function in a specialized replication process required to repair double strand breaks (16). In addition to replicative and repair roles, it has been suggested that pol ⑀ coordinates transcriptional and cell cycle responses to DNA damage and replication blocks (17).In S. cerevisiae, a BRCT domain-containing protein, Dpb11, interacts with the pol ⑀ complex and was originally identified as a suppressor of pol ⑀ catalytic and Dpb2 subunit mutants (18,19). DPB11 is an essential gene required for DNA replication (18). The inability of DPB11 mutants to restrain mitosis in the presence of inco...

Background: Replicative DNA polymerases ␦ and ⑀ are believed to synthesize lagging and leading strands, respectively. Results: Human DNA polymerases ␣/␦ and ⑀ segregate during S phase and DNA polymerase ⑀ alone remains bound to lamins. Conclusion: DNA polymerases ␦ and ⑀ act independently in late S phase Significance: Human cell DNA replication may mechanistically differ from prokaryotic replication.

Lactic acid bacteria (LAB) are known as predominant beer spoilers. They cause turbidity, acidity, gas formation and offflavors in beer by formation of side metabolites. Beer spoiling LAB have a substantial financial impact in the brewing industry making their rapid detection and identification essential. Despite the developed rapid diagnostic methods, the bottleneck in detection remains the lengthy enrichment cultivation step. This paper describes the applicability of a novel glucose auto delivery system, EnBase™, for the improved enrichment cultivation of beer spoiling LAB in MRS medium. By means of the applied system, glucose is slowly released into the culture during growth, which results in faster enrichment. Growth of Lactobacillus brevis DSM 20054 T and several beer spoiling LAB was accelerated resulting in up to a 300% increase in the cell density after 48 h of cultivation compared to the commonly used MRS medium. A test of naturally contaminated beer samples indicated that the addition of glucose by means of EnBase allows faster detection of LAB in breweries.

RNA polymerase II (RNA pol II) transcribes proteinencoding genes in eukaryotes. It can be purified as a 'core' enzyme containing 10-12 subunits with a molecular mass of 500 kDa. However, larger RNA pol II-containing complexes, capable of transcribing from model promoters in vitro with minimal addition of general transcription factors, have also been purified [1]. These 'holoenzyme' complexes contain general transcription factors, other transcriptional mediators, as well as various sets of accessory proteins, such as chromatin remodelling factors [2]. The carboxy-terminal domain (CTD) of RNA pol II has been implicated in mediating interactions with other factors involved in transcription and mRNA processing, and appears to be a major target of regulation. The CTD comprises tandem heptapeptide repeats of the DNA polymerase e co-operates with polymerases a and d in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase e and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase e was also shown to UV crosslink specifically a-amanitin-sensitive transcripts, unlike DNA polymerase a that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase e, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase e in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase e complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.Abbreviations BrdU, bromodeoxyuridine; CTD, carboxyterminal domain; DRB, 5,6-dichloro-1-beta-D-ribobenzimidazole; Pol, DNA polymerase; RNA pol II, RNA polymerase II; TFIIH, transcription factor II H.