Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.Myocyte development involves the coordinated activation of many genes. Aspects of this regulation have been elucidated by the identification of nuclear proteins that specifically bind to muscle gene DNA regulatory elements and by the isolation of myogenic determination factors. A number of conserved factor-binding sites have been identified in the promoters and enhancers of muscle-specific genes. The sequence motif CANNTG, or E-box, occurs in the musclespecific creatine kinase and myosin light-chain 1/3 genes (8,18). This motif is the binding site for myogenic determination factors and other proteins that share a conserved helix-loophelix motif (3,24,29,30). Another conserved sequence motif, CATTCCT, or MCAT, occurs in a variety of muscle gene promoters and is essential for expression from the cardiac troponin T promoter (25). A ubiquitous protein has been identified that specifically binds to MCAT motifs from the chicken cardiac troponin T and the skeletal actin promoters (26). The CC(A/T)6GG motif, or CArG, occurs in single or multiple copies in the promoters of the sarcomeric actin genes as well as in the nonmuscle actins, interleukin receptor, and c-fos genes (27,32,42). The proximal CArG motif in the chicken skeletal actin promoter forms the core of a muscle regulatory element (MRE) that is sufficient for muscle-specific expression upstream from a TATA element (44). In contrast, the CArG motif in the c-fos promoter forms the core of the serum response element (SRE), which confers rapid and transient activation of expression in response to serum growth factors and is required for basal, constitutive expression in the nonmuscle actin genes (28,43). DNA elements that contain the CArG motif interact...

DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specffic expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK's essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2DA746 frameshift reversion assay, we show DDK is required to generate "complex" spontaneous mutations, which are a hallmark of the Polz translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polz. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polz may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polzdependent mutagenesis, DDK was also important for generating Polz-independent large deletions that revert the lys2DA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7D pol32D and cdc7D pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication "gap filling" rather than during "polymerase switching" by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32. K NOWLEDGE of how mutations are produced is important for understanding genetic variability in populations and the process of evolution. Cells have evolved sophisticated molecular mechanisms for maintaining the integrity of the genome. Most DNA repair mechanisms have high fidelity and prevent mutations by removing damaged DNA and replacing the resulting gap using the undamaged, complementary strand as a template (Waters et al. 2009;Boiteux and Jinks-Robertson 2013). If not repaired, some lesions block the replicative DNA polymerases (Pols a, d, and e) during S phase and require bypass by an alternative errorfree or error-prone mechanism. Error-free bypass usually involves a template switch to the undamaged sister chromatid, while error-prone bypass uses translesion synthesis (TLS) DNA polymerases to synthesize new DNA directly across the lesion. There are at least 15 identified TLS polymerases in human cells but only three in the yeast Saccharomyces cerevisiae: Polz, Polh, and Rev1, which are encoded by the REV3-REV7, RAD30, and REV1 genes, respectively (Boiteux and Jinks-Robertson 2013). TLS polymerases have low fidelity and processivity on undamaged DNA templates and lack an associated exonuclease proofreading activity.The misregulation or loss of TLS p...

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