Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD, RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD, RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10 -100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase .Mutagenesis caused by UV light and by many other DNA damaging agents in Escherichia coli is under control of the SOS response, a highly regulated stress response, which functions to increase cell survival under adverse environmental conditions that cause DNA damage (1). Genetic analysis has uncovered four genes, whose products are required for SOS mutagenesis. Two of these, DNA polymerase III (pol-III) 1 and RecA, participate also in replication and recombination, respectively.The other two, UmuD and UmuC, are specifically required for the mutagenic reaction. It was found that UmuD is processed into a shorter form, UmuDЈ, which is the form active in SOS mutagenesis (reviewed in Ref.2).Based on in vivo and in vitro data, UmuDЈ and UmuC were thought to be accessory proteins, which assist DNA polymerase III in replicating DNA lesions which usually block replication (2-5). According to this mechanism, the mutations occur by misinsertion opposite the DNA lesion by the DNA polymerase, a result of the miscoding nature of most DNA lesions. Recently SOS mutagenesis was reconstituted with purified components in two laboratories (6, 7). The results, which confirmed an earlier study (4), provided strong biochemical evidence that SOS mutagenesis occurs by replication through DNA lesions, in a reaction which depends on UmuC, UmuDЈ, RecA and SSB. Moreover, it was shown that there is a qualitative difference in the specificity of bypass when translesion replication was compared in the absence or presence of SOS proteins. DNA polymerase III holoenzyme bypassed an abasic site via a misalignment mechanism, resulting in skipping over the lesion, and the formation of Ϫ1 frameshifts (7,8). In contrast, in the p...

Upon DNA damage signaling, p73, a member of the p53 tumor suppressor family, accumulates to support transcription of downstream apoptotic genes. p73 interacts with Yes-associated protein 1 (Yap1) through its PPPY motif, and increases p73 transactivation of apoptotic genes. The ubiquitin E3 ligase Itch, like Yap1, interacts with p73. Given the fact that both Itch and Yap1 bind p73 via the PPPY motif, we hypothesized that Yap may also function to stabilize p73 by displacing Itch binding to p73. We show that the interaction of Yap1 and p73 was necessary for p73 stabilization. Yap1 competed with Itch for binding to p73, and prevented Itch-mediated ubiquitination of p73. Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of Yap1 siRNA. Altogether, our findings attribute a central role to Yap1 in regulating p73 accumulation and function under DNA damage signaling.

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin-dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. Alternatively, certain proteins are inherently unstable and undergo "default" degradation by the 20S proteasomes. Puzzlingly, proteins are by large subjected to both degradation pathways. Proteins with unstructured regions have been found to be substrates of the 20S proteasomes in vitro and, therefore, unstructured regions may serve as signals for protein degradation "by default" in the cell. The literature is loaded with examples where engagement of a protein into larger complexes increases protein stability, possibly by escaping degradation "by default". Our model suggests that formation of protein complexes masks the unstructured regions, making them inaccessible to the 20S proteasomes. This model not only provides molecular explanations for a recent theoretical "cooperative stability" principle, but also provokes new predictions and explanations in the field of protein regulation and functionality.

The rapid synthesis and breakdown ofmRNA in prokaryotes can impose a significant energy drain on these while RNA turnover in Escherichia colU was hydrolytic, it was nonhydrolytic in Bacillus sublilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. colU extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase H activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nudeoside diphosphates as products. A low level of a Mn21-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coil and B. sublilis extracts differ by about 20-to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed.RNA accounts for about 20o of the dry mass of a bacterial cell (1). Depending on the growth rate of the cell, as much as 50-60o of the RNA synthesized at any one time can be messenger RNA (mRNA) (2). Inasmuch as mRNA accumulates to only a few percent of the total cellular RNA (1), it is clear, as has been known for many years, that mRNA must be rapidly degraded. As a consequence of these considerations, a bacterial cell expends a significant amount ofenergy in maintaining the pool of mRNA responsible for gene expression.Despite the large amount of work in recent years devoted to understanding the mechanisms and specificity of mRNA breakdown in vivo (3, 4), relatively little attention has been given to the energetics ofthe process, particularly to whether the energy state of the cell may influence the mode of mRNA decay. In an early series of studies, Boyer and coworkers (5, 6) suggested, on the basis of 18' labeling patterns, that mRNA breakdown in Escherichia coli is predominantly hydrolytic, whereas in Bacillus subtilis it is predominantly nonhydrolytic. However, the enzymatic basis for the divergence between these two organisms was not established at that time.Based on studies with E. coli mutants, it is now known that the final breakdown of mRNA to the mononucleotide level in vivo is dependent on the enzymes RNase II and polynucleotide phosphorylase (PNPase) (7-10). Cells lacking only one of these enzymes are viable, whereas the absence of both enzymes leads to inviability and the accumulation of mRNA fragments (9,10). RNase II and PNPase have similar modes of action (11,12). Both are processive exoribonucleases initiating breakdown at the 3' terminus of an RNA molecule, and both are influenced by secondary structure in the RNA. Howeve...

Error-prone DNA repair consists of replicative filling-in of DNA gaps carrying lesions. We have reconstituted E. coli SOS error-prone repair using purified DNA polymerase III holoenzyme, SSB, RecA, UmuD', a UmuC fusion protein, and a gap lesion plasmid. In the absence of UmuDC, or without SOS induction, replication skips over the lesion, forming mostly one-nucleotide deletions. These cause translational frameshifts that usually inactivate genes. UmuD' and UmuC, in the presence of RecA and SSB, stimulate translesion replication and change its mutagenic specificity such that deletions are prevented and base substitutions are increased. This results in mutagenic but nondetrimental gap repair and provides an effective mechanism for generating genetic variation in bacteria adapting to environmental stress.

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 533 exonuclease that shares homology with Red␣, commonly known as exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Red␤ synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl 2 , 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA (dsDNA) virus with a 152-kb linear genome. Replication of HSV-1 DNA takes place in the host nucleus. The first step of viral replication involves the circularization of the genome (13, 43). Shortly thereafter, replication intermediates appear as longer-than-unit-length head-to-tail concatemers (19) that have undergone genomic inversion (2, 27, 47, 58). The genome concatemers are not linear but rather consist of a mixture of complex structures such as Y-and X-shaped branches, replication bubbles, and tangled masses (20,28,48,49). The presence of these structures and the inversion of the L and S genome segments suggest that recombination plays a role in the replication of HSV-1 DNA. In fact, high levels of recombination are known to accompany HSV infection (3,10,11,46,54). While cellular recombinases may be involved in mediating some of these processes (57), the possibility exists that HSV-1 encodes recombinases that can also participate.Herpes simplex virus type 1 (HSV-1) encodes a 5Ј-to-3Ј exonuclease (17, 23, 32, 52) termed alkaline nuclease, the product of the UL12 open reading frame (29). Recently, computer database searches have revealed that the HSV-1 UL12 gene shares homology with bacteriophage lambda Red␣, commonly known as exonuclease (1, 36). The Red␣ protein is a 5Ј33Ј exonuclease which is part of the Red recombinase previously shown to be required for recombination by bacteriophage lambda in a RecA Ϫ host (8,9,50). Red␣ operates in conjunction with a single-stranded DNA (ssDNA) binding protein, lambda Red␤, which promotes ssDNA annealing (34). The lambda Red recomb...

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.