Rad26p, a yeast homologue of human Cockayne syndrome B with an ATPase activity, plays a pivotal role in stimulating DNA repair at the coding sequences of active genes. On the other hand, DNA repair at inactive genes or silent areas of the genome is not regulated by Rad26p. However, how Rad26p recognizes DNA lesions at the actively transcribing genes to facilitate DNA repair is not clearly understood in vivo. Here, we show that Rad26p associates with the coding sequences of genes in a transcription-dependent manner, but independently of DNA lesions induced by 4-nitroquinoline-1-oxide in Saccharomyces cerevisiae. Further, histone H3 lysine 36 methylation that occurs at the active coding sequence stimulates the recruitment of Rad26p. Intriguingly, we find that Rad26p is recruited to the site of DNA lesion in an elongating RNA polymerase II-dependent manner. However, Rad26p does not recognize DNA lesions in the absence of active transcription. Together, these results provide an important insight as to how Rad26p is delivered to the damage sites at the active, but not inactive, genes to stimulate repair in vivo, shedding much light on the early steps of transcription-coupled repair in living eukaryotic cells.

RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.

Recently, we have demonstrated a predominant association of Rad26p with the coding sequences but not promoters of several GAL genes following transcriptional induction. Here, we show that the occupancy of histone H2A–H2B dimer at the coding sequences of these genes is not altered following transcriptional induction in the absence of Rad26p. A histone H2A–H2B dimer-enriched chromatin in Δrad26 is correlated to decreased association of RNA polymerase II with the active coding sequences (and hence transcription). However, the reduced association of RNA polymerase II with the active coding sequence in the absence of Rad26p is not due to the defect in formation of transcription complex at the promoter. Thus, Rad26p regulates the occupancy of histone H2A–H2B dimer, which is correlated to the association of elongating RNA polymerase II with active GAL genes. Similar results are also found at other inducible non-GAL genes. Collectively, our results define a new role of Rad26p in orchestrating chromatin structure and hence transcription in vivo.

No abstract

Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability.

Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.The genomic DNA is continuously attacked by numerous damaging agents (1). The cellular lesions interfere with various types of DNA transacting processes, leading to extensive degenerative diseases (2-5). However, the integrity of the genomic DNA is protected from genotoxic factors by an intricate network of mechanisms in both prokaryotic and eukaryotic organisms (6 -13). The most versatile cellular mechanism to deal with a large variety of DNA lesions is nucleotide excision repair, which primarily removes helix-distorting lesions, including 4-nitroquinoline-1-oxide (4NQO) 3 -induced bulky chemical adducts. Oxidative lesions and small base alterations are removed by base excision repair (8), whereas the doublestranded DNA breaks are processed by homology-dependent recombination or DNA end-joining (9, 10).A severe ramification of DNA damage is the formation of lesions in the transcribing strands of the active genes. These lesions cause the distortion of local helical DNA structure, pausing the elongating RNA polymerase II (14 -18), thereby leading to a transcriptional arrest. Such a transcriptional blockage interferes with cell function or triggers apoptosis (19, 20). Fortunately, the cell employs a specific repair mechanism to efficiently remove lesions from the transcribing strands of the active genes for normal cellular activities. Such a repair mode is referred to as transcription-coupled DNA repair (6, 21-25). Transcription-coupled DNA repair is present in both prokaryotic and eukaryotic organisms (26). In prokaryotes, transcription repair coupling factor displaces the stalled RNA polymerase, facilitating recruitment of the DNA repair machinery to the lesion. However, the mechanisms, regulation, and intricate connections of the events involved in eukaryotic transcriptioncoupled DNA repair are considerably more complex and poorly understood.Several studies (16,(27)(28)(29) in eu...

Rtt109p, a histone acetyltransferase, associates with active genes and acetylates lysine 56 on histone H3 in Saccharomyces cerevisiae. However, the functional role of Rtt109p or H3 Lys 56 acetylation in chromatin assembly/disassembly (and hence gene expression) immediately switching transcription on or off has not been clearly elucidated in vivo. Here, we show that Rtt109p promotes the eviction of histone H3 from a fast inducible yeast gene, GAL1, following transcriptional initiation via histone H3 Lys 56 acetylation. Conversely, the deposition of histone H3 to GAL1 is significantly decreased in the presence of Rtt109p following transcriptional termination. Intriguingly, we also find that the deposition of histone H2B on preexisting non-acetylated histone H3 Lys 56 at GAL1 in ⌬rtt109 is significantly increased independently of histone H3 deposition immediately following transcriptional termination subsequent to a short induction. Consistently, histone H2B is not efficiently evicted from GAL1 in the absence of Rtt109p immediately following transcriptional induction. Furthermore, we show that the stimulated eviction or reduced deposition of histones by Rtt109p promotes the association of RNA polymerase II with GAL1 and hence the synthesis of GAL1 mRNA. These results, taken together, support the fact that Rtt109p regulates the deposition/ eviction of histone H2B in addition to its role in stimulating histone H3 eviction, thus providing insight into chromatin assembly/disassembly and hence gene expression in vivo.The post-translational covalent modifications of histones play important roles in DNA transacting processes, such as transcription, replication, and DNA repair (1-7). Most of the covalent modifications occur at the N-terminal tails of histones with the exception of ubiquitylation that occurs at the C-terminal tails of histones H2A and H2B. However, covalent modifications also occur in the globular core domain. One such modification is acetylation of lysine 56 on histone H3 (8, 9). This modification has been speculated to play an important role in the regulation of histone H3-DNA interaction within the nucleosome because it is present at the entry and exit sites of nucleosomal DNA (8, 10, 11). Histone H3 Lys 56 acetylation functions in promoting cell survival following exposure to genotoxic agents (9). Further, this covalent modification has been implicated in error-free replication (12, 13), gene activation (8, 11), RNA polymerase II transcript elongation through heterochromatin (14), and the pathogenesis of Candida albicans (15). Thus, histone H3 Lys 56 acetylation is an important covalent modification that regulates transcription, replication, DNA repair, and pathogenesis (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19).To identify the factor(s) or enzyme (s) (16 -20) and shares structural homology with metazoan p300/CREB 4 -binding protein (21-23). In fact, Das et al. (24) have recently demonstrated the role of human p300/CREB-binding protein in histone H3 Lys 56 acetylation. Rtt109p requires either of two ...

Background:To determine whether DNA double-strand break (DSB) repair is coupled to transcription, we analyzed DSB repair at the active and inactive genes. Results: Our results reveal that DSB repair at the active gene is faster than that at the inactive gene. Conclusion:These results demonstrate a preferential DSB repair at the active gene. Significance: This study supports the existence of transcription-coupled DSB repair.