SummaryLesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.

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Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI͞SNF family of ATPases͞ helicases, and it possesses a C 3HC4 RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6 -Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.postreplication repair ͉ translesion synthesis ͉ tumor suppressor

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.Genomic instability underlies the development of various human diseases, including cancer. Cancer genomes are highly heterogeneous and can possess various instability phenotypes, including accumulation of gross chromosomal rearrangements (GCR) (9, 40). A plethora of evidence has indicated that defects in various DNA repair pathways promote genomic instability and trigger subsequent tumor formation (1). One example is the mutational or epigenetic inactivation of the mismatch repair gene MSH2 or MLH1 in a subset of colorectal cancers (4, 39). An apparently distinct subset of colon cancers, representing about 40% of malignant colorectal transformations, is characterized by epigenetic inactivation of the HLTF gene (22). While HLTF has been suggested to act as a transcription factor (31), recent studies indicated a role for HLTF in replication of damaged DNA, raising the possibility that it is this function of HLTF that can lead to suppression of genomic instability (23, 37).In Saccharomyces cerevisiae, genetic data have indicated a crucial role for the Rad6-Rad18 protein complex, Rad5, and the Mms2-Ubc13 complex in the replication of damaged DNA (26). In contrast to nucleotide incorporation opposite the lesion by specialized translesion synthesis polymerases, which requires Rad6-Rad18-dependent monoubiquitylation of PCNA, template-switching-mediated bypass depends on Lys63 polyubiquitylation of PCNA by the Mms2-Ubc13 ubiquitin-conjugating enzyme complex and Rad5 ubiquitin ligase (10,12,36,41). Yeast genetic data have shown that not only the ubiquitin ligase activity of Rad5 but also its ATPase activity is essential for its function in replication of damaged DNA (5). In agreement with the in vivo data, Rad5 is an ATP-hydrolysis-driven molecular motor which can facilitate template switching at stalled replication forks (2). While translesi...

SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core-and PBAPselective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.Gene expression control is one of the most fundamental biological processes and, to a large extent, occurs at the transcriptional level. The transcription of a single protein-encoding eukaryotic gene involves a stunning plethora of regulating factors comprising some 100 or so distinct polypeptides (16,23,28). These can be classified as sequence-specific DNA-binding transcription factors that initiate the recruitment of positive or negative coregulatory complexes and the basal transcription machinery. Coactivators include a variety of proteins performing distinct functions during the transcription cycle such as the opening up of chromatin structure, mediating posttranslational histone modifications or bridging between activators and the basal transcription machinery. It has become clear that the diversity among gene-specific activators and repressors is complemented by functional specification among coregulatory complexes and even the core transcription machinery. One important class of coregulators is formed by the ATP-dependent chromatin-remodeling factors (remodelers).Remodelers are large multisubunit complexes defined by the presence of an ATPase "engine" subunit (2, 3, 21, 24, 32). These proteins act like DNA translocases and use the energy derived from ATP hydrolysis to change the DNA-histone contacts, thus remodeling chromatin structure (4, 41). Based on the identity of their central ATPase, four major classes of remodelers have been recognized: SWI/SNF, ISWI, CHD/Mi2, and Ino80/Swr1 (17). Different remodelers are not exchangeable; rather, each executes unique biological functions. An early example of functional diversification was our finding that the Drosophila SWI/SNF class Brahma (BRM) remodelers, but not the ISWI remodelers, act as chromatin-specific coac...

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. Here we show that the grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, we propose that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity.In Drosophila, the bithorax complex (BX-C) is responsible for providing the identity of segments posterior to the mesothorax. This homeotic gene complex contains only three coding genes, while the major part of BX-C is arranged into a complex array of functionally autonomous, segment-specific cis-regulatory elements. During early development, the transiently expressed segmentation gene products establish the active or inactive states of the cis regulators, which is then maintained by the trithorax group (trx-G) and the Polycomb group (Pc-G) of genes, respectively, during the rest of development (31). The protein products of the trx-G and Pc-G genes are recruited as distinct protein complexes to the regulatory elements by specific target sites, called trithorax and Polycomb response elements (TRE and PRE, respectively) (12,20,51,56).Among the known PREs, the iab-7 PRE is particularly well characterized. In transgenic constructs, the iab-7 PRE induces silencing of reporter genes (23). Silencing is stronger when the transgene is homozygous, a phenomenon observed with most PREs and commonly referred to as pairing-sensitive silencing (PSS) (30). PSS by the iab-7 PRE depends on the products of Pc-G, as mutations in several members of this group, including Scm, Pc, Ph, Sce, Psc, z, etc., relieve PSS (10,23). Five out of the six above-mentioned genes encode proteins that together form the functional core of the PRC1 protein complex, whereas SCM only substoichiometrically associates with it (18, 49). Transgenic constructs carrying the iab-7 PRE create a new binding site for PC, PSC, and PH, and PC was also shown to bind to the iab-7 PRE in vivo and in situ (10,45). These data indicate that a P...

SummaryKip-related proteins (KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin-dependent kinase (CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt) by a yeast two-hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two-hybrid test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D-type cyclins. However, in the pull-down assays, B-type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded weakly to this inhibitor protein. Kinase complexes from G 2 /M cells showed increased sensitivity towards the inhibitor compared with those isolated from G 1 /S-phase cells. In vitro phosphorylation of Medicago retinoblastoma-related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin-like domain protein kinase (MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin-dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross-talk between Ca 2þ signalling and regulation of cell-cycle progression in plants.

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.