Nucleotide excision repair and photolyase repair of UV photoproducts ...

337

REVIEW / SYNTHE`SE

Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivo1 Maxime Tremblay, Martin Toussaint, Annie D’Amours, and Antonio Conconi

Abstract: The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA. Key words: chromatin, cyclobutane pyrimidine dimer, nucleosome, nucleotide excision repair, photolyase, psoralen crosslinking, rDNA, ribosomal genes, RNA polymerase I, UV. Re´sume´ : Le geńome est organise´ en sous-domaine nucleáire, creánt des re´gions de chromatine diffe´rentes tant au niveau fonctionnel que structurel (e.g., se´quences hautement re´pe´titives, centrome`res, te´lome`res, se´quences non-codantes, ge`nes inactifs, ge`nes transcrits par l’ARN polyme´rase II et III, et le nucleóle). Il existe une corre´lation entre la proximite´ d’un ` l’oppose´, les ge`nes encodant l’ARN riboge`ne a` une re´gion he´te´rochromatique et son niveau d’activite´ transcriptionnel. A somal (ARNr), qui sont transcrits a` un tre`s fort taux (~60 % de la transcription totale d’une cellule), posse`dent une structure de chromatine ouverte et sont contenus dans le nucleóle. De plus, ces ge`nes d’ARNr existent sous 2 formes; les ge`nes actifs ne posse`dent aucun nucleósome, alors que les ge`nes inactifs sont compacte´s par les nucleósomes. Tout comme la transcription et la re´plication de l’ADN, la re´paration est aussi moduleé par la structure de la chromatine. Bien que la re´paration geńe´rale de l’ADN total soit d’une importance primordiale pour comprendre les mećanismes de maintien du geńome, cette revue de litte´rature cible principalement la re´paration par excision de nucleótide et la re´version directe par la photolyase des dommages creé´s par les UV, et ce, spećifiquement dans l’e´le´ment structurel de la chromatine : le nucleósome. De plus, il y est pre´sente´ un re´sume´ des e´tudes de´montrant l’existence des 2 types de chromatine des ge`nes encodant l’ARNr, et comment cette particularite´ permet une comparaison directe de la re´paration de l’ADN dans 2 structures tre`s diffe´rentes de chromatine (nucleósomale et non-nucleósomale). Mots-cle´s : chromatine, dime`re cyclobutylique de pyrimidine, nucleósome, re´paration par excision de nucleótides, photolyase, psorale`ne, photopontage, ADNr, ge`nes ribosomaux, ARN polyme´rase I, UV. [Traduit par la Re´daction]

Received 26 June 2008. Revision received 28 July 2008. Accepted 29 July 2008. Published on the NRC Research Press Web site at bcb.nrc.ca on 13 February 2009. M. Tremblay, M. Toussaint, A. D’Amours, and A. Conconi.2 De´partement de Microbiologie et Infectiologie, Faculte´ de Me´decine, Universite´ de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. 1This

paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process. 2Corresponding author (e-mail: [email protected]). Biochem. Cell Biol. 87: 337–346 (2009)

doi:10.1139/O08-128

Published by NRC Research Press

338

UV-induced DNA damage and chromatin Environmental agents, such as the ultraviolet (UV) component of sunlight, ionizing radiation, and numerous genotoxic chemicals and pollutants, cause DNA damage. If not repaired, DNA damage can lead to mutations and increased risk for cancer (Hoeijmakers 2001). UV light induces 2 major types of damage: cyclobutane pyrimidine dimers (CPDs), which cause 70%–80% of total damage; and (6-4) pyrimidine-pyrimidone dimers ((6-4)PDs), which cause 20%–30% of total damage. CPDs are formed by covalent bonds between 2 carbon atoms (C-5 and C-6) of adjacent pyrimidines in the same DNA strand, forming a cyclobutyl ring; (6-4)PDs are formed by a covalent bond between the C-6 position of one pyrimidine and the C-4 position of the adjacent pyrimidine. Both photoproducts, but in particular the (6-4)PDs, cause a significant helical distortion and bending in DNA (Friedberg et al. 2006). Although, neither photoproducts have a major effect on the stability of the higherorder structure of chromatin, DNA bending can promote changes in the interactions between DNA and proteins (e.g., transcription factors), or can modify the nucleosome translational and rotational settings. The extent to which photoproducts affect the stability of nucleosomes depends on the DNA sequence (reviewed in Smerdon and Conconi 1999). The repeating unit of chromatin fibers, the nucleosome, contains an octamer of the 4 core histone proteins (H2A, H2B, H3, and H4) and ~168 bp of DNA coiled in 2 lefthanded turns around the octamer surface (Luger et al. 1997). The core particles are connected by a variable length (0–60 bp) of linker DNA in complex with linker histones (H1 or H5). Different classes of DNA lesions form preferentially in linker DNA or about equally (per unit DNA) in linker and core regions (reviewed in Smerdon and Conconi 1999). For instance, CPDs form almost randomly between linker and nucleosome cores, whereas (6-4)PDs form preferentially in linker DNA (Mitchell et al. 1990; Niggli and Cerutti 1982; Suquet et al. 1995). Although there is little bias for CPD formation between nucleosome core and linker DNA, the distribution of CPDs within the core DNA is significantly modulated (Gale et al. 1987; Gale and Smerdon 1988, 1990). In fact, CPDs form with an average periodicity of 10.3 bases (UV photofootprint), reflecting the rotational setting of DNA on the surface of the histone octamer, similar to DNase I digestion footprints (Noll 1974). Thus, CPDs are introduced most easily near positions in the DNA helix that are far from the histone surface. However, it is not the presence of histones but the bending of DNA around the octamer that promotes the UV photofootprinting of nucleosomes (Brown et al. 1993; Pehrson and Cohen 1992). This is explained by the formation of CPDs that widen the minor groove, whereas the minor groove in nucleosome cores is compressed toward the histone surface (reviewed in Smerdon and Conconi 1999). Contrary to the distinct distribution of CPDs, the formation of (6-4)PDs within the nucleosome core is almost random (Gale and Smerdon 1990).

Nucleotide excision repair in nucleosomes Nucleotide excision repair (NER) is the pathway that removes UV photoproducts from the DNA. It is performed by a large multienzymatic complex, or complexes, made of

Biochem. Cell Biol. Vol. 87, 2009

more than 30 proteins, that repairs DNA via distinct steps: recognition of the lesion, incision of the damaged DNA strand upstream and downstream of the lesion, excision of the resulting ~30 nt DNA fragment containing the lesion, filling the gap by DNA synthesis, and ligation of the newly synthesized patch (Fig. 1A) (Friedberg et al. 2006). NER is divided in 2 subpathways: global genome repair (GG-NER) and transcription-coupled repair (TC-NER). GG-NER repairs transcription inactive DNA and the nontranscribed strand (NTS) of active genes, whereas TC-NER repairs the transcribed strand (TS) of active genes (Hoeijmakers 2001; Mellon et al. 1987; Mellon and Hanawalt 1989; Smerdon and Thoma 1990; Tornaletti and Hanawalt 1999; Verhage et al. 1998). Most of the NER proteins participate in both subpathways, except for some of those involved in the DNA damage recognition step (Table 1). In general, TC-NER is more efficient than GG-NER, and elongation by RNA polymerase (RNAP)II is required for TC-NER (Bohr et al. 1985; Mellon et al. 1986). The links between transcription and fast repair are not well understood, but RNA polymerases stalled at damaged sites could be the signal for the fast recruitment of repair proteins (Donahue et al. 1994; Hara et al. 1999; Park et al. 2002; Sarker et al. 2005). Then, stalled RNA polymerases may be displaced or backed up to make DNA lesions accessible to the large NER complex (Sarker et al. 2005; Tornaletti and Hanawalt 1999; Mellon 2005). In human cells, the product of the CSB gene has been implicated in the recognition and displacement of stalled RNA polymerases (reviewed in Laine´ and Egly 2006). At first, it was thought that TC-NER of UV-induced photolesions occurred exclusively in RNAPII-transcribed genes. However, more recently, TC-NER has been also associated with RNAPI transcription, at least in budding yeast (Verhage et al. 1996; Conconi et al. 2002, 2005a; Meier et al. 2002; Tremblay et al. 2008). The enzymatic complex of NER is largely conserved from yeast to human (Table 1), and studies in yeast have made major contributions in elucidating the mechanisms of NER in humans (reviewed in Prakash and Prakash 2000). The strength of the yeast system is its reliance on both genetic and biochemical approaches, which combined make a powerful tool with which to study intricate nuclear processes, such as DNA transcription, replication, recombination, and repair. The NER enzymes must recognize DNA lesions, remove them, and synthesize a DNA patch in the different nuclear domains and chromatin structures. Therefore, understanding the modulation of NER by chromatin, as well as the changes occurring in the structure of chromatin during NER, is crucial to our comprehension of the fate of potential mutagenic and carcinogenic lesions in DNA. Smerdon and Lieberman pioneered this research field with a series of elegant experiments using human fibroblasts, which were done to investigate the distribution of unscheduled DNA synthesis (DNA synthesis step; Fig. 1A) in nucleosomes (Lan and Smerdon 1985; Jensen and Smerdon 1990), the rearrangement (unfolding or sliding) of nucleosomes during NER (Smerdon and Lieberman 1978), the formation of nucleosomes (refolding or sliding) after repair, and the repositioning of nucleosomes on the repaired regions (Hunting et al. 1985; Smerdon 1986; Smerdon et al. 1979; Smerdon and LieberPublished by NRC Research Press

Tremblay et al.

339

Fig. 1. (A) Nucleotide excision repair (NER) steps. UV photoproducts induce a kink in the DNA, and the DNA damage is recognized. After DNA unwinding, single-strand incisions are made at both sides of the lesion. An oligonucleotide (~30 nt) containing the lesion is excised, and the resulting gap is filled (repair synthesis; grey arrow), using the opposite DNA strand as template. The newly synthesized strand is ligated by DNA ligase. (B) Model of NER in the nucleosome. Recognition of DNA lesion is followed by rearrangement of the nucleosome (sliding or unfolding), which gives access to NER enzymes to the lesion. Repair DNA synthesis and ligation are followed by repositioning of the nucleosome on the newly synthesized patch (light grey region).

man 1980). Among the important results are those showing that, during the DNA synthesis step, the newly repaired DNA is not tightly bound to the surface of core histones and, thus, that the structure of nucleosomes is altered during NER. In addition, it was found that the DNA ligation step precedes nucleosome formation (Smerdon 1986). This set of experiments led the authors to propose the working model shown in Fig. 1B. To study the complexity of NER in chromatin, researchers have used in vitro repair reactions. For instance, early studies showed that human cell extracts could not efficiently repair UV-irradiated plasmid DNA when it was reconstituted in nucleosomes (Wang et al. 1991), and that DNA repair was more effective in naked DNA than in SV40 minichromosomes (Sugasawa et al. 1993). In another study (Araki et al. 2000), purified NER factors were added to SV40 minichromosomes. It was found that NER was slower in SV40 minichromosomes than in naked control DNA. Under different experimental conditions, a positioned nucleosome was reconstituted on a randomly damaged 175-bplong DNA fragment containing the Xenopus borealis somatic 5S rRNA gene, and Xenopus oocyte nuclear extracts were used to carry out repair. These experiments showed that nucleosomes strongly inhibited NER at many CPD sites. Moreover, removal of histone tails from the core histones had little effect on the repair rates (Liu and Smerdon 2000). In parallel, a short DNA fragment (165 bp), containing a single CPD (designed to be positioned 5 bases from the dyad center), was reconstituted in a nucleosome. The NER activity present in Xenopous nuclear extracts repaired the

CPD, albeit at half of the rate at which the CPD was repaired in naked DNA (Kosmoski et al. 2001). Similarly, NER was followed in a mononucleosome containing 1 (6-4)PD at a defined position. Using either cell extracts or reconstituted human excision nuclease factors, it was found that the nucleosome reduced the affinity for DNA of the XPA, RPA, and XPC proteins, which are involved in damage recognition. Consequently, core DNA was repaired at a rate corresponding to ~10% of that measured in naked DNA (Hara et al. 2000). Surprisingly, the excision of (6-4)PD was also inhibited when present in linker DNA between reconstituted dinucleosomes (Ura et al. 2001). In summary, these studies show that nucleosomes, the firstorder packing of DNA in chromatin, determine the repair rate, at least in vitro. Since the early works on NER in chromatin using cultured human cells (reviewed in Smerdon and Conconi 1999), important progress has also been made in vivo. Studies in yeast have shown that DNA sequences occupied by nucleosomes are repaired slowly. Moreover, removal of CPDs and (6-4)PDs from the TS of 2 active genes is fast and uniform, while removal of both photoproducts from the NTS is generally less efficient, and is modulated by the position of the nucleosomes (Wellinger and Thoma 1997; Tijsterman et al. 1999; Ferreiro et al. 2004). Yet, it is unclear why only the repair rate in the NTS is modulated by the presence of nucleosomes (Teng et al. 2005). On another level of DNA packing, NER was followed in a yeast heterochromatin-like structure, which contains silent information regulator (Sir) proteins, and forms on genes that are posiPublished by NRC Research Press

340


Table 1. Nucleotide excision repair (NER) proteins in yeast and human. NER protein

Function

Saccharomyces cerevisiæ Rad1/10p ssDNA endonuclease; cleaves 5’ of the damage Rad2p ssDNA endonuclease; cleaves 3’ of the damage Rad3p Subunit of TFIIH; 5’–3’ helicase Rad4p Damaged DNA binding protein Rad14p Damaged DNA binding protein Rad7/16p-Abf1 ATP-dependant binding to damaged DNA; involved in GG-NER Rad23p Binds/regulates Rad4p Rad26p DNA-dependant ATPase activity; involved in RNA polymerase II TC-NER Rad28p Unknown Rad33p Binds Rad4p Rad34p Binds Rad23p; belongs to Rad4p/XPC family Tfb1p TFIIH subunit Tfb2p TFIIH subunit Tfb4p TFIIH subunit, interact with Ssl1p Ssl1p TFIIH subunit, interact with Tfb4p Ssl2p (RAD25) Subunit of TFIIH; 3’–5’ helicase Met18p (MMS19) Regulates TFIIH activity RPA (Rfa1/2/3p) ssDNA binding Human XPF-ERCC1 XPG XPD XPC XPA DDB1 DDB2

HR23B CSB CSA Centrin2 p62 p52 p34 p44 XPB hMMS19 RPA

ssDNA endonuclease; cleaves 5’ of the damage ssDNA endonuclease; cleaves 3’ of the damage Subunit of TFIIH; 5’–3’ helicase Damaged DNA binding protein; role suggested in TFIIH positioning Damaged DNA binding protein DDB1-CUL4A-based ligase Participates in ubiquitylation of histones H3 and H4; facilitates the cellular response to DNA damage Binds to XPC DNA-dependant ATPase activity; involved in RNA polymerase II TC-NER Binds to DDB1; involved in RNA polymerase II TC-NER Binds XPC and stimulates the NER pathway TFIIH subunit TFIIH subunit TFIIH subunit TFIIH subunit Subunit of TFIIH; 3’–5’ helicase Interacts with XPD and XPB ssDNA binding

Note: ss, single stranded; GG-NER, global genome repair nucleotide excision repair; TC-NER, transcription-coupled repair nucleotide excision repair; TFIIH, transcription factor II H; DDB1, DNA-damage-binding protein.

tioned near the telomeres. CPD removal was studied in a URA3 gene inserted 2 kb from the telomere, and it was found that NER is inhibited in the coding region, at the promoter and 3’ end, when the gene is fully silenced (LivingstoneZatchej et al. 2003). The large size of the NER complex intuitively calls for rearrangement of nucleosomes during DNA repair. Therefore, histone modifications and chromatin remodeling could play important roles, for instance, by promoting the accessibility of the DNA to NER enzymes and (or) helping restore the original structure of chromatin on the repaired DNA. Indeed, there is evidence that covalent modifications of his-

tones take place during NER (Ramanathan and Smerdon 1989; Teng et al. 2002; Yu et al. 2005) and that chromatin remodeling promotes NER (Citterio et al. 2000; Ura et al. 2001; Hara and Sancar 2002; Frit et al. 2002; Hara and Sancar 2003; Yu et al. 2005; Gong et al. 2006; Nag et al. 2008; Green and Almouzni 2002; Ura and Hayes 2002). Also, the chromatin assembly factor (CAF)-1, which has a central role in replication-dependent chromatin assembly (Smith and Stillman 1989; reviewed in Verreault 2000), appears to assist in restoring the original structure of chromatin after DNA repair (Gaillard et al. 1996, 1997; Moggs et al. 2000; Mello et al. 2002; Mello and Almouzni 2001; Green and Almouzni 2003). All these recent studies have refined the working-model shown in Fig. 1B (see Green and Almouzni 2002). It is noteworthy, however, that, in the promoter of the yeast MFA2 gene, hyperacetylation of histone H3 and chromatin remodeling occur within minutes of UV irradiation, and in the absence of NER (Yu et al. 2005). Moreover, UV photoproducts induce chromatin assembly in the active ribosomal genes in the absence of NER (see Chromatin and the repair of UV photoproducts in ribosomal genes). These results suggest that some chromatin modifications could occur independent of NER. Thus, despite evidence that chromatin modification facilitates DNA repair, it is not clear how much this process is needed for efficient NER in nucleosomes (Hara et al. 2000; Bucceri et al. 2006; Thoma 2005). In fact, the intrinsic mobility of nucleosomes observed in vitro and in cells could allow enough free space for NER to access DNA damage. Since chromatin remodeling during DNA repair is not the main focus of this report, we refer the reader to more comprehensive reviews on this subject (Ura and Hayes 2002; Waters and Smerdon 2005; Groth et al. 2007).

Photolyase repair in nucleosomes An alternative mechanism to repair UV photoproducts is photoreactivation, which directly reverses pyrimidine dimers to their monomeric form. This process is catalyzed by an enzyme called photolyase, which is present in several (but not all) species from each of the 3 kingdoms (reviewed in Sancar 2003). Photolyases are monomeric proteins (55–65 kDa) of 2 different types: CPD photolyases and (6-4)PD photolyases (Carell et al. 2001). In Escherichia coli, the globularshaped CPD photolyase contains 2 chromophore cofactors: a flavin adenine dinucleotide and a methenyltetrahydrofolate. The repair mechanism of photoreactivation is well known: photolyase binds to DNA containing a CPD in a lightindependent reaction, the CPD flips into the active site pocket, catalysis is initiated by light (350–450 nm), and then the CPD is split into 2 pyrimidines (Kao et al. 2005; Mees et al. 2004). At the end, the enzyme dissociates from the repaired DNA (Weber 2005). Importantly, binding of photolyase induces major DNA bending, with an average angle of 368 (van Noort et al. 1999). Thus, DNA wrapping around nucleosome cores could interfere with photoreactivation by restraining the formation of optimal DNA bending. In yeast, photolyase preferentially repairs the NTS of RNAPII-transcribed (Livingstone-Zatchej et al. 1997; Suter et al. 1997) and RNAPIII-transcribed genes (Aboussekhra and Thoma 1998); this is not true for RNAPI-transcribed genes (Meier et al. 2002). Therefore, it is suggested that Published by NRC Research Press

Tremblay et al.

yeast RNAPII and RNAPIII blocked at CPDs inhibits the access of photolyase to DNA lesions, as previously shown by in vitro experiments (Donahue et al. 1994). Like NER, photolyase must recognize pyrimidine dimers from a large excess of nondimerized pyrimidine doublets, a process that is aggravated by the structure of chromatin (reviewed by Thoma 1999). A series of reports have shown that photolyase rapidly repairs CPDs in non-nucleosome regions (e.g., promoters of active genes and linker DNA), while nucleosomes restrict its accessibility to DNA lesions. More precisely, slow repair occurs in the center of nucleosomes, with a gradual increase in the repair rate toward the periphery. Thus, repair of CPDs by photolyase is tightly modulated by the structure of chromatin. Interestingly, nucleosomes modulate photoreactivation of CPDs, even in the TS of active genes, which is in contrast to the uniform removal of CPDs by NER in the same DNA strand of active genes (see above) (Suter et al. 1997, 2000a, 2000b; Morse et al. 2002; Suter and Thoma 2002). Furthermore, there is a clear correlation between the patterns obtained after MNase digestion of chromatin (an in vitro assay) and repair of CPDs by photolyase in yeast. Consequently, photolyase can be used as a molecular tool to study chromatin structure in vivo (Suter et al. 1999). (Note that nucleosomes also strongly inhibit photoreactivation in vitro. In these experiments, photoreactivation was tested using reconstituted nucleosomes and E. coli DNA photolyase (Schieferstein and Thoma 1998; Kosmoski and Smerdon 1999; Kosmoski et al. 2001; Gaillard et al. 2003).)

Chromatin and the repair of UV photoproducts in ribosomal genes How modifications of histone proteins and chromatin remodeling affect NER in vivo is a challenging question. To address it, NER is measured in mutant cells that miss one or more chromatin remodeling factors. However, these cell lines often present changes in the transcriptome. Consequently, it is difficult to discriminate if putative changes in DNA repair kinetics result from a modified chromatin structure or if they are the indirect consequence of global changes in gene expression. Therefore, most of the current knowledge has been gained by in vitro studies, where NER has been followed in reconstituted chromatin, in the presence or absence of chromatin remodeling factors, and compared with NER in naked DNA controls. We selected the yeast ribosomal genes (rRNA genes or rDNA) to help answer some of the questions related to NER in chromatin and chromatin remodeling in vivo. The attractive feature of the rDNA locus is its chromatin structure, which is well characterized. Specifically, not all rDNA is transcribed, and both active rRNA genes (absence of canonical nucleosomes) and inactive rRNA genes (presence of canonical nucleosomes) coexist in the cell. These characteristics allow for direct comparison of NER in 2 very different chromatin structures, in which the non-nucleosome rDNA fraction is the intrinsic control (reviewed in Conconi 2005). The following paragraph summarizes most of the studies that have contributed to the demonstration of the existence of 2 distinct rDNA chromatins, what is known about NER and photolyase repair in the yeast rDNA locus, and how these

341

2 DNA repair mechanisms assess the presence of nucleosome and non-nucleosome rDNA. In eukaryotic cells, rRNA genes are present in multiple copies of 2 distinct types: one is permissive to transcription and the other is transcriptionally refractive (reviewed in Grummt and Pikaard 2003). Thus, rRNA synthesis can be regulated by 3 different mechanisms: designation of the total number of active genes, modulation of the transcription initiation rate, and regulation of the RNAPI elongation rate (reviewed in Grummt 1999; Moss 2004, 2007; Conconi et al. 1989; Stefanovsky et al. 2006). In Saccharomyces cerevisiae, rRNA synthesis appears to be regulated by varying the proportions of active and inactive rDNA copies in response to growth conditions, and by modulating the transcription initiation rate of active genes, which is controlled by the enhancer DNA element (Dammann et al. 1993; Banditt et al. 1999; reviewed in Reeder 1999). More recently, it has been proposed that the overall initiation rate, but not the number of active genes, determines the rRNA transcription rate during exponential growth (French et al. 2003). Another study supports the possibility that modulation in rRNA synthesis is the result of 2 events: a change in transcription rate and a small but significant change in the number of active rDNA copies (Fahy et al. 2005). However, this work has also shown that variation in transcription rates of active genes is the most significant event, in agreement with French et al. (2003). At the chromatin level, canonical nucleosomes are not present in the coding regions of rRNA genes that are active. Conversely, nucleosomes are found on the same coding DNA sequences when genes are inactive. Similar to silent rDNA, nucleosomes are present on most of the DNA sequences flanking the rRNA genes (reviewed in Lucchini and Sogo 1998; Sogo and Thoma 2003). These characteristics have been observed in a variety of organisms, ranging from yeast (Dammann et al. 1993, 1995; Smith and Boeke 1997; Smith et al. 1999) to microplasmodia (Lucchini et al. 1987), slime mold (Sogo et al. 1984), plants (Conconi et al. 1992), insects (Sanz et al. 2007), amphibians (Lucchini and Sogo 1992), and mammals (Conconi et al. 1989; Fritz and Smerdon 1995; Stancheva et al. 1997), using a technique based on psoralen photocrosslinking (reviewed in Toussaint et al. 2005). Recently, it has been reported that in a yeast strain with a reduced number of rDNA repeats (~40 copies instead of the ~150 copies present in the wild-type strains), there are unphased nucleosomes on the active rRNA genes (Jones et al. 2007). This conclusion, which was drawn from micrococcal nuclease digestion and chromatin immunoprecipitation assays, relied on the belief that in yeast with reduced numbers of rDNA repeats, all rRNA genes must be active. Subsequently, however, Merz et al. (2008) demonstrated that this is not the case, and that 10%–20% of rDNA is inactive in the yeast strain carrying just 40 copies of rRNA genes. Moreover, the authors further showed that active rDNA is free of nucleosomes by chromatin endogenous cleavage (Merz et al. 2008; Schmid et al. 2004). Finally, electron micrographs of psoralen-crosslinked rDNA chromatin clearly show that nucleosomes are not present on the coding regions of active ribosomal genes of yeast (Dammann et al. 1993), Dictyostelium discoideum (Sogo et al. 1984; De Bernardin et al. 1986), Physarum polycephalum Published by NRC Research Press

342

(Lucchini et al. 1987), or of a mouse cell line (J.M. Sogo 2004, personal communication). These studies corroborate a number of biochemical analyses (accessibility of micrococcal nuclease, DNase I, and restriction enzymes, together with psoralen crosslinking band-shifts), which also support the existence of a non-nucleosome structure for active rDNA (e.g., Ness et al. 1983; Sogo et al. 1984; Lucchini et al. 1987; Lucchini and Sogo 1992; Cavalli et al. 1996; Muller et al. 2000; Conconi et al. 2005a). This is a reasonable conclusion, considering that rRNA genes are transcribed at a high elongation rate and at maximal density of RNA polymerases: ~1 RNAPI per 100 base pairs (SollnerWebb and Mougey 1991; Grummt 1999) (or ~2 RNAPI per nucleosome DNA). Most studies on DNA repair have focused on the genome overall or on RNAPII-transcribed genes in particular. However, a number of studies have also analyzed DNA repair in the nucleolus (reviewed in Conconi et al. 2005b). For example, the activities of NER and photolyase repair were followed in the rDNA locus of yeast. It was found that CPDs are not repaired from rDNA in rad1, 2, 3, and 14 yeast mutants. These findings have demonstrated that CPDs in the highly repetitive rDNA arrays are mostly removed by NER, not by other repair mechanisms, such as homologous recombination (Verhage et al. 1996). Moreover, the same study proposed that NER in the nucleolus is DNA-strand specific. Later, the existence of TC-NER dependent on RNAPI transcription was investigated by measuring repair in the TS and NTS of active rDNA, and compared with measurements taken in the inactive rDNA. These studies have shown that TC-NER occurs in active rDNA, but not in inactive rDNA (Conconi et al. 2002; Meier et al. 2002). Interestingly, repair of CPDs from the NTS of active rDNA is faster than from either strand of inactive rDNA, indicating that active rDNA chromatin is repaired more efficiently than inactive rDNA chromatin (Conconi et al. 2002, 2005a). Since nucleosomes delay NER, this result underscores the presence of non-nucleosome and nucleosome rDNA units in yeast (Tremblay et al. 2008). The activity of photolyase is strongly modulated by the presence of nucleosomes, and measurements of photoreversal of CPDs give a very good indication of whether defined DNA sequences are folded in nucleosomes. As anticipated, photolyase repairs active rDNA considerably faster than inactive rDNA (Meier et al. 2002). In addition, photolyase repairs, at similar rates, inactive rDNA and the GAL10 gene, which is packaged in nucleosomes when cells are grown in glucose (Cavalli and Thoma 1993). Finally, repair of CPDs by photolyase was used to investigate the structure of chromatin in the rDNA promoter and in the DNA sequences flanking the rRNA genes (spacer DNA) (Meier and Thoma 2005). Under those experimental conditions, nucleosome-free DNA is repaired in ~15 min (e.g., in the nuclease-sensitive promoter region), whereas DNA in nucleosomes is repaired in ~2 h (e.g., spacer DNA). Again, the repair kinetics of spacer DNA and inactive rDNA coding regions were very similar. Therefore, repair of CPDs by photolyase substantiates the existence of both nucleosome and non-nucleosome rDNA units. Little is known about the way NER proceeds through open (non-nucleosome) chromatin. Initial information was obtained by analyzing NER in the yeast rDNA locus (Con-


coni et al. 2002; Meier et al. 2002). It was found that within minutes after UV irradiation, chromatin reorganizes over the non-nucleosome rDNA units. Importantly, chromatin assembly on the active rDNA occurred in the absence of DNA repair. Thus, UV-induced DNA lesions blocked rDNA transcription and triggered the formation of an inactive chromatin structure. The resumption of transcription after removal of CPDs correlated with the reappearance of non-nucleosome rDNA chromatin (Conconi et al. 2005a). However, at present, it is unclear whether the displacement of RNAPI stalled at CPDs promotes the formation of inactive rDNA chromatin, or whether nucleosomes assemble at, and propagate from, the damaged sites before repair is completed, as was found in reconstituted repair experiments in vitro (Gaillard et al. 1997; Moggs et al. 2000).

Conclusion Several studies in vitro and in vivo have clearly shown that nucleosomes (and other protein–DNA complexes) are an impediment to repair mechanisms, such as NER and photoreactivation. This is true for the genome overall, as well as for specific nuclear domains, such as centromeres, subtelomeric regions, inactive and active genes that are transcribed by RNAPII and RNAPIII, and the inactive rDNA units. Conversely, NER and photoreactivation are generally efficient in nucleosome-free regions, such as linker DNA and the active rDNA units. However, how cells detect and remove DNA lesions in chromatin is still unknown, and it remains a very challenging and, likely, a long-standing question. Another important question, which is largely unanswered, is how histone modification and chromatin remodeling participate in DNA repair. The yeast rDNA locus, with its active and inactive structures, has been successfully used as a biological model to study chromatin during DNA transcription and replication. Thus, it is possible that it could be used to help elucidate a number of open questions about DNA repair in chromatin.

Acknowledgments We thank Dr. K. Kobryn (Universite´ de Sherbrooke) for critical reading of the manuscript. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A.C.

References Aboussekhra, A., and Thoma, F. 1998. Nucleotide excision repair and photolyase preferentially repair the nontranscribed strand of RNA polymerase III-transcribed genes in Saccharomyces cerevisiae. Genes Dev. 12: 411–421. doi:10.1101/gad.12.3.411. PMID:9450934. Araki, M., Masutani, C., Maekawa, T., Watanabe, Y., Yamada, A., Kusumoto, R., et al. 2000. Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins. Mutat. Res. 459: 147–160. PMID:10725665. Banditt, M., Koller, T., and Sogo, J.M. 1999. Transcriptional activity and chromatin structure of enhancer-deleted rRNA genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 4953–4960. PMID:10373545. Bohr, V.A., Smith, C.A., Okumoto, D.S., and Hanawalt, P.C. 1985. DNA repair in an active gene: removal of pyrimidine dimmers Published by NRC Research Press

Tremblay et al. from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell, 40: 359–369. doi:10.1016/00928674(85)90150-3. PMID:3838150. Brown, D.W., Libertini, L.J., Suquet, C., Small, E.W., and Smerdon, M.J. 1993. Unfolding of nucleosome cores dramatically changes the distribution of ultraviolet photoproducts in DNA. Biochemistry, 32: 10527–10531. doi:10.1021/bi00091a001. PMID:8399198. Bucceri, A., Kapitza, K., and Thoma, F. 2006. Rapid accessibility of nucleosomal DNA in yeast on a second time scale. EMBO J. 25: 3123–3132. doi:10.1038/sj.emboj.7601196. PMID:16778764. Carell, T., Burgdorf, L.T., Kundu, L.M., and Cichon, M. 2001. The mechanism of action of DNA photolyases. Curr. Opin. Chem. Biol. 5: 491–498. doi:10.1016/S1367-5931(00)00239-8. PMID:11578921. Cavalli, G., and Thoma, F. 1993. Chromatin transitions during activation and repression of galactose-regulated genes in yeast. EMBO J. 12: 4603–4613. PMID:8223470. Cavalli, G., Bachmann, D., and Thoma, F. 1996. Inactivation of topoisomerases affects transcription-dependent chromatin transitions in rDNA but not in a gene transcribed by RNA polymerase II. EMBO J. 15: 590–597. PMID:8599942. Citterio, E., Van Den Boom, V., Schnitzler, G., Kanaar, R., Bonte, E., Kingston, R.E., et al. 2000. ATP-dependent chromatin remodeling by the Cockayne syndrome B DNA repair-transcriptioncoupling factor. Mol. Cell. Biol. 20: 7643–7653. doi:10.1128/ MCB.20.20.7643-7653.2000. PMID:11003660. Conconi, A. 2005. The yeast rDNA locus: a model system to study DNA repair in chromatin. DNA Repair (Amst.), 4: 897–908. doi:10.1016/j.dnarep.2005.04.008. PMID:15996904. Conconi, A., Widmer, R.M., Koller, T., and Sogo, J.M. 1989. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell, 57: 753–776. doi:10.1016/00928674(89)90790-3. PMID:2720786. Conconi, A., Sogo, J.M., and Ryan, C.A. 1992. Ribosomal gene clusters are uniquely proportioned between open and closed chromatin structures in both tomato leaf cells and exponentially growing suspension cultures. Proc. Natl. Acad. Sci. U.S.A. 89: 5256–5260. doi:10.1073/pnas.89.12.5256. PMID:11607297. Conconi, A., Bespalov, V., and Smerdon, M.J. 2002. Transcription coupled repair in RNA polymerase I transcribed genes of yeast. Proc. Natl. Acad. Sci. U.S.A. 99: 649–654. doi:10.1073/pnas. 022373099. PMID:11782531. Conconi, A., Paquette, M., Fahy, D., Bespalov, V.A., and Smerdon, M.J. 2005a. Repair-independent chromatin assembly onto active ribosomal genes in yeast after UV irradiation. Mol. Cell. Biol. 25: 9773–9783. doi:10.1128/MCB.25.22.9773-9783.2005. PMID: 16260595. Conconi, A., Bespalov, V.A., Fahy, D., and Smerdon, J.M. 2005b. DNA repair in RNA Polymerase-I transcribed genes. In From DNA photolesions to mutations, skin cancer and cell death. Edited by R. Drouin, E. Sage, and M. Rouabhia. RSC Publishing. Cambridge, U.K. pp. 123–147. Dammann, R., Lucchini, R., Koller, T., and Sogo, J.M. 1993. Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae. Nucleic Acids Res. 21: 2331–2338. doi:10. 1093/nar/21.10.2331. PMID:8506130. Dammann, R., Lucchini, R., Koller, T., and Sogo, J.M. 1995. Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences. Mol. Cell. Biol. 15: 5294–5303. PMID:7565678. De Bernardin, W., Koller, T., and Sogo, J.M. 1986. Structure of invivo transcribing chromatin as studied in simian virus 40 mini-

343 chromosomes. J. Mol. Biol. 191: 469–482. doi:10.1016/00222836(86)90142-7. PMID:3029385. Donahue, B.A., Yin, S., Taylor, J.-S., Reines, D., and Hanawalt, P.C. 1994. Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimmer in the DNA template. Proc. Natl. Acad. Sci. U.S.A. 91: 8502–8506. doi:10.1073/pnas. 91.18.8502. PMID:8078911. Fahy, D., Conconi, A., and Smerdon, M.J. 2005. Rapid changes in transcription and chromatin structure of ribosomal genes in yeast during growth phase transitions. Exp. Cell Res. 305: 365–373. doi:10.1016/j.yexcr.2005.01.016. PMID:15817161. Ferreiro, J.A., Powell, N.G., Karabetsou, N., Kent, N.A., Mellor, J., and Water, R. 2004. Cbf1p modulates chromatin structure, transcription and repair at the Saccharomyces cerevisiae MET16 locus. Nucleic Acids Res. 32: 1617–1626. doi:10.1093/nar/ gkh324. PMID:15007107. French, S.L., Osheim, Y.N., Cioci, F., Nomura, M., and Beyer, A.L. 2003. In exponentially growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes. Mol. Cell. Biol. 23: 1558–1568. doi:10.1128/MCB.23.5.15581568.2003. PMID:12588976. Friedberg, E.C., Wood, R.D., Walker, G.C., Schultz, R.A., Siede, W., and Ellenberger, T. 2006. DNA repair and mutagenesis. 2nd ed. ASM Press, Washington, DC. Frit, P., Kwon, K., Coin, F., Auriol, J., Dubaele, S., Salles, B., and Egly, J.M. 2002. Transcriptional activators stimulate DNA repair. Mol. Cell, 10: 1391–1401. doi:10.1016/S1097-2765(02) 00732-3. PMID:12504014. Fritz, L.K., and Smerdon, M.J. 1995. Repair of UV damage in actively transcribed ribosomal genes. Biochemistry, 34: 13117– 13124. doi:10.1021/bi00040a024. PMID:7548072. Gaillard, P.-H.L., Martini, E.M.-D., Kaufman, P.D., Stillman, B., Moustacchi, E., and Almouzni, G. 1996. Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I. Cell, 86: 887–896. doi:10.1016/S0092-8674(00)80164-6. PMID:8808624. Gaillard, P.-H.L., Moggs, J.G., Roche, D.M.J., Quivy, J.-P., Becker, P.B., Wood, R.D., and Almouzni, G. 1997. Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair. EMBO J. 16: 6281–6289. doi:10.1093/emboj/16.20.6281. PMID:9321407. Gaillard, H., Fitzgerald, D.J., Smith, C.L., Peterson, C.L., Richmond, T.J., and Thoma, F. 2003. Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase. J. Biol. Chem. 278: 17655– 17663. doi:10.1074/jbc.M300770200. PMID:12637512. Gale, J.M., and Smerdon, M.J. 1988. Photofootprint of nucleosome core DNA in intact chromatin having different structural states. J. Mol. Biol. 204: 949–958. doi:10.1016/0022-2836(88)90054-X. PMID:3221402. Gale, J.M., and Smerdon, M.J. 1990. UV induced (6-4) photoproducts are distributed differently than cyclobutane dimers in nucleosomes. Photochem. Photobiol. 51: 411–417. doi:10.1111/j. 1751-1097.1990.tb01732.x. PMID:2160660. Gale, J.M., Nissen, K.A., and Smerdon, M.J. 1987. UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases. Proc. Natl. Acad. Sci. U.S.A. 84: 6644–6648. doi:10.1073/pnas.84.19.6644. PMID:3477794. Gong, F., Fahy, D., and Smerdon, M.J. 2006. Rad4-Rad23 interaction with SWI/SNF links ATP-dependent chromatin remodeling with nucleotide excision repair. Nat. Struct. Mol. Biol. 13: 902– 907. doi:10.1038/nsmb1152. PMID:17013386. Published by NRC Research Press

344 Green, C.M., and Almouzni, G. 2002. When repair meets chromatin. First in series on chromatin dynamics. EMBO Rep. 3: 28– 33. doi:10.1093/embo-reports/kvf005. PMID:11799057. Green, C.M., and Almouzni, G. 2003. Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo. EMBO J. 22: 5163–5174. doi:10.1093/emboj/cdg478. PMID:14517254. Groth, A., Roccha, W., Verreault, A., and Almouzni, G. 2007. Chromatin challenges during DNA replication and repair. Cell, 128: 721–733. doi:10.1016/j.cell.2007.01.030. PMID:17320509. Grummt, I. 1999. Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog. Nucleic Acid Res. Mol. Biol. 62: 109–154. doi:10.1016/S0079-6603(08)60506-1. PMID:9932453. Grummt, I., and Pikaard, C.S. 2003. Epigenetic silencing of RNA polymerase I transcription. Nat. Rev. Mol. Cell Biol. 4: 641– 649. doi:10.1038/nrm1171. PMID:12923526. Hara, R., and Sancar, A. 2002. The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle. Mol. Cell. Biol. 22: 6779–6787. doi:10.1128/MCB.22.19.6779-6787.2002. PMID:12215535. Hara, R., and Sancar, A. 2003. Effect of damage type on stimulation of human excision nuclease by SWI/SNF chromatin remodelling factor. Mol. Cell. Biol. 23: 4121–4125. doi:10.1128/ MCB.23.12.4121-4125.2003. PMID:12773556. Hara, R., Shelby, C.P., Liu, M., Price, D.H., and Sancar, A. 1999. Human transcription release factor 2 dissociates RNA polymerases I and II stalled at a cyclobutane thymine dimer. J. Biol. Chem. 274: 24779–24786. doi:10.1074/jbc.274.35.24779. PMID:10455150. Hara, R., Mo, J., and Sancar, A. 2000. DNA damage in the nucleosome core is refractory to repair by human excision nuclease. Mol. Cell. Biol. 20: 9173–9181. doi:10.1128/MCB.20.24.91739181.2000. PMID:11094069. Hoeijmakers, J.H. 2001. Genome maintenance mechanism for preventing cancer. Nature, 411: 366–374. doi:10.1038/35077232. PMID:11357144. Hunting, D.J., Dresler, S.L., and Lieberman, M.W. 1985. Multiple conformational states of repair patches in chromatin during DNA excision repair. Biochemistry, 24: 3219–3226. doi:10. 1021/bi00334a022. PMID:3927974. Jensen, K.A., and Smerdon, J.M. 1990. DNA repair within nucleosome cores of UV-irradiated human cells. Biochemistry, 29: 4773–4782. doi:10.1021/bi00472a005. PMID:2364058. Jones, H.S., Kawauchi, J., Braglia, P., Alen, C.M., Kent, N.A., and Proudfoot, N.J. 2007. RNA polymerase I in yeast transcribes dynamic nucleosomal rDNA. Nat. Struct. Mol. Biol. 14: 123–130. doi:10.1038/nsmb1199. PMID:17259992. Kao, Y.T., Saxena, C., Wang, L., Sancar, A., and Zhong, D. 2005. Direct observation of thymine dimer repair in DNA by photolyase. Proc. Natl. Acad. Sci. U.S.A. 102: 16128–16132. doi:10. 1073/pnas.0506586102. PMID:16169906. Kosmoski, J.V., and Smerdon, M.J. 1999. Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site. Biochemistry, 38: 9485–9494. doi:10.1021/bi990297h. PMID:10413526. Kosmoski, J.V., Ackerman, E.J., and Smerdon, M.J. 2001. DNA repair of a single UV photoproduct in a designed nucleosome. Proc. Natl. Acad. Sci. U.S.A. 98: 10113–10118. doi:10.1073/ pnas.181073398. PMID:11517308. Laine´, J.P., and Egly, J.M. 2006. When transcription and repair meet: a complex system. Trends Genet. 22: 430–436. doi:10. 1016/j.tig.2006.06.006. PMID:16797777. Lan, S.Y., and Smerdon, J.M. 1985. A nonuniform distribution of

Biochem. Cell Biol. Vol. 87, 2009 excision repair synthesis in nucleosome core DNA. Biochemistry, 24: 7771–7783. doi:10.1021/bi00347a041. PMID:4092038. Liu, X., and Smerdon, M.J. 2000. Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome. J. Biol. Chem. 275: 23729–23735. doi:10.1074/jbc.M002206200. PMID:10821833. Livingstone-Zatchej, M., Meier, A., Suter, B., and Thoma, F. 1997. RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes. Nucleic Acids Res. 25: 3795–3800. doi:10.1093/nar/25.19.3795. PMID:9380500. Livingstone-Zatchej, M., Marcionelli, R., Mo¨ller, K., de Pril, R., and Thoma, F. 2003. Repair of UV lesions in silenced chromatin provides in vivo evidence for a compact chromatin structure. J. Biol. Chem. 278: 37471–37479. doi:10.1074/jbc.M306335200. PMID:12882973. Lucchini, R., and Sogo, J.M. 1992. Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes. Mol. Cell. Biol. 12: 4288–4296. PMID:1406621. Lucchini, R., and Sogo, J.M. 1998. The dynamic structure of ribosomal RNA gene chromatin. In Transcription of ribosomal RNA genes by eukaryotic RNA polymerase I. Edited by M.R. Paule. M.R. R.G. Landes Company, Austin, Texas. pp. 254–276. Lucchini, R., Pauli, U., Braun, R., Koller, T., and Sogo, J.M. 1987. Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum. J. Mol. Biol. 196: 829–843. doi:10. 1016/0022-2836(87)90408-6. PMID:3681980. Luger, K., Ma¨der, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature, 389: 251–260. doi:10.1038/ 38444. PMID:9305837. Mees, A., Klar, T., Gnau, P., Hennecke, U., Eker, A.P., Carell, T., and Essen, L.O. 2004. Crystal structure of a photolyase bound to a CPD-like DNA lesion after in situ repair. Science, 306: 1789– 1793. doi:10.1126/science.1101598. PMID:15576622. Meier, A., and Thoma, F. 2005. RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair. Mol. Cell. Biol. 25: 1586– 1595. doi:10.1128/MCB.25.5.1586-1595.2005. PMID:15713619. Meier, A., Livingstone-Zatchej, M., and Thoma, F. 2002. Repair of active and silenced rDNA in yeast. J. Biol. Chem. 277: 11845– 11852. doi:10.1074/jbc.M110941200. PMID:11805105. Mello, J.A., and Almouzni, G. 2001. The ins and outs of nucleosome assembly. Curr. Opin. Genet. Dev. 11: 136–141. doi:10. 1016/S0959-437X(00)00170-2. PMID:11250135. Mello, J.A., Sillje´, H.H., Roche, D.M., Kirschner, D.B., Nigg, E.A., and Almouzni, G. 2002. Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway. EMBO Rep. 3: 329–334. doi:10.1093/embo-reports/kvf068. PMID:11897662. Mellon, I. 2005. Transcription-coupled repair: a complex affair. Mutat. Res. 577: 155–161. PMID:15913669. Mellon, I., and Hanawalt, P.C. 1989. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature, 342: 95–98. doi:10.1038/342095a0. PMID:2554145. Mellon, I., Bohr, V.A., Smith, C.A., and Hanawalt, P.C. 1986. Preferential DNA repair of an active gene in human cells. Proc. Natl. Acad. Sci. U.S.A. 83: 8878–8882. doi:10.1073/pnas.83.23. 8878. PMID:3466163. Mellon, I., Spivak, G., and Hanawalt, P.C. 1987. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell, 51: 241–249. doi:10.1016/0092-8674(87)90151-6. PMID:3664636. Published by NRC Research Press

Tremblay et al. Merz, K., Hondele, M., Goetze, H., Gmelch, K., Stoeckl, U., and Griesenbeck, J. 2008. Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules. Genes Dev. 22: 1190–1204. doi:10. 1101/gad.466908. PMID:18451108. Mitchell, D.L., Nguyen, T.D., and Cleaver, J.E. 1990. Non-random induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin. J. Biol. Chem. 265: 5353– 5356. PMID:2318816. Moggs, J.G., Grandi, P., Quivy, J.P., Jonsson, Z.O., Hubscher, U., Becker, P.B., and Almouzni, G. 2000. A CAF-1–PCNAmediated chromatin assembly pathway triggered by sensing DNA damage. Mol. Cell. Biol. 20: 1206–1218. doi:10.1128/ MCB.20.4.1206-1218.2000. PMID:10648606. Morse, N.R., Meniel, V., and Waters, R. 2002. Photoreactivation of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene of Saccharomyces cerevisiae. Nucleic Acids Res. 30: 1799– 1807. doi:10.1093/nar/30.8.1799. PMID:11937634. Moss, T. 2004. At the crossroads of growth control; making ribosomal RNA. Curr. Opin. Genet. Dev. 14: 210–217. doi:10.1016/j. gde.2004.02.005. PMID:15196469. Moss, T., Langlois, F., Gagnon-Kugler, T., and Stefanovsky, V. 2007. A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell. Mol. Life Sci. 64: 29–49. doi:10. 1007/s00018-006-6278-1. PMID:17171232. Muller, M., Lucchini, R., and Sogo, J.M. 2000. Replication of yeast rDNA initiates downstream of transcriptionally active genes. Mol. Cell, 5: 767–777. doi:10.1016/S1097-2765(00)80317-2. PMID:10882113. Nag, R., Gong, F., Fahy, D., and Smerdon, M.J. 2008. A single amino acid change in histone H4 enhances UV survival and DNA repair in yeast. Nucleic Acids Res. 36: 3857–3866. PMID:18508805. Ness, P.J., Labhart, P., Banz, E., Koller, T., and Parish, R.W. 1983. Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation. J. Mol. Biol. 166: 361–381. doi:10.1016/S0022-2836(83) 80090-4. PMID:6304325. Niggli, H.J., and Cerutti, P. 1982. Nucleosomal distribution of thymine photodimers following far- and near-ultraviolet irradiation. Biochem. Biophys. Res. Commun. 105: 1215–1223. doi:10. 1016/0006-291X(82)91098-1. PMID:7092895. Noll, M. 1974. Internal structure of the chromatin subunit. Nucleic Acids Res. 1: 1573–1578. doi:10.1093/nar/1.11.1573. PMID:10793712. Park, J.S., Marr, M.T., and Roberts, J.W. 2002. E. coli transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation. Cell, 109: 757–767. doi:10. 1016/S0092-8674(02)00769-9. PMID:12086674. Pehrson, J.R., and Cohen, L.H. 1992. Effects of DNA looping on pyrimidine dimer formation. Nucleic Acids Res. 20: 1321–1324. doi:10.1093/nar/20.6.1321. PMID:1561089. Prakash, S., and Prakash, L. 2000. Nucleotide excision repair in yeast. Mutat. Res. 451: 13–24. PMID:10915862. Ramanathan, B., and Smerdon, M.J. 1989. Enhanced DNA repair synthesis in hyperacetylated nucleosomes. J. Biol. Chem. 264: 11026–11034. PMID:2738057. Reeder, R.H. 1999. Regulation of RNA polymerase I transcription in yeast and vertebrates. Prog. Nucleic Acid Res. Mol. Biol. 62: 293–327. doi:10.1016/S0079-6603(08)60511-5. PMID:9932458. Sancar, A. 2003. Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem. Rev. 103: 2203– 2237. doi:10.1021/cr0204348. PMID:12797829.

345 Sanz, C., Gorab, E., Ruiz, M.F., Sogo, J.M., and Dıéz, J.L. 2007. Chromatin structure of ribosomal genes in Chironomus thummi (Diptera: Chironomidae): tissue specificity and behaviour under drug treatment. Chromosome Res. 15: 429–438. doi:10.1007/ s10577-007-1134-1. PMID:17487564. Sarker, A.H., Tsutakawa, S.E., Kostek, S., Ng, C., Shin, D.S., Peris, M., et al. 2005. Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne Syndrome. Mol. Cell, 20: 187–198. PMID:16246722. Schieferstein, U., and Thoma, F. 1998. Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V in vitro. EMBO J. 17: 306– 316. doi:10.1093/emboj/17.1.306. PMID:9427764. Schmid, M., Durussel, T., and Laemmli, U.K. 2004. ChIC and ChEC; genomic mapping of chromatin proteins. Mol. Cell, 16: 147–157. PMID:15469830. Smerdon, M.J. 1986. Completion of excision repair in human cells. Relationship between ligation and nucleosome formation. J. Biol. Chem. 261: 244–252. PMID:3941074. Smerdon, M.J., and Conconi, A. 1999. Modulation of DNA damage and DNA repair in chromatin. Prog. Nucleic Acid Res. Mol. Biol. 62: 227–255. doi:10.1016/S0079-6603(08)60509-7. PMID:9932456. Smerdon, M.J., and Lieberman, M.W. 1978. Nucleosome rearrangement in human chromatin during UV-induced DNA-repair synthesis. Proc. Natl. Acad. Sci. U.S.A. 75: 4238–4241. doi:10. 1073/pnas.75.9.4238. PMID:279912. Smerdon, M.J., and Lieberman, M.W. 1980. Distribution within chromatin of deoxyribonucleic acid repair synthesis occurring at different times after ultraviolet radiation. Biochemistry, 19: 2992–3000. doi:10.1021/bi00554a025. PMID:7397113. Smerdon, M.J., and Thoma, F. 1990. Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell, 61: 675–684. doi:10.1016/0092-8674(90)90479-X. PMID:2188732. Smerdon, M.J., Kastan, M.B., and Lieberman, M.W. 1979. Distribution of repair-incorporated nucleotides and nucleosome rearrangement in the chromatin of normal and xeroderma pigmentosum human fibroblasts. Biochemistry, 18: 3732–3739. doi:10.1021/bi00584a014. PMID:476082. Smith, J.S., and Boeke, J.D. 1997. An unusual form of transcriptional silencing in yeast ribosomal DNA. Genes Dev. 11: 241– 254. doi:10.1101/gad.11.2.241. PMID:9009206. Smith, J.S., Caputo, E., and Boeke, J.D. 1999. A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors. Mol. Cell. Biol. 19: 3184–3197. PMID:10082585. Smith, S., and Stillman, B. 1989. Purification and characterization of CAF-1, a human cell factor required for chromatin assembly during DNA replication. Cell, 58: 15–25. doi:10.1016/00928674(89)90398-X. PMID:2546672. Sogo, J.M., and Thoma, F. 2003. The structure of rDNA chromatin. In The nucleolus. Edited by M.O.J. Olson. Kluwer Academic/ Plenum Publishers. New York. pp. 1–15. Sogo, J.M., Ness, P.J., Widmer, R.M., Parish, R.W., and Koller, T. 1984. Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin. J. Mol. Biol. 178: 897–919. doi:10.1016/0022-2836(84)90318-8. PMID:6092647. Sollner-Webb, B., and Mougey, E.B. 1991. News from the nucleolus: rRNA gene expression. Trends Biochem. Sci. 16: 58–62. doi:10.1016/0968-0004(91)90025-Q. PMID:1858134. Stancheva, I., Lucchini, R., Koller, T., and Sogo, J.M. 1997. Chromatin structure and methylation of rat rRNA genes studied by Published by NRC Research Press

346 formaldehyde fixation and psoralen cross-linking. Nucleic Acids Res. 25: 1727–1735. doi:10.1093/nar/25.9.1727. PMID:9108154. Stefanovsky, V., Langlois, F., Gagnon-Kugler, T., Rothblum, L.I., and Moss, T. 2006. Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling. Mol. Cell, 21: 629– 639. doi:10.1016/j.molcel.2006.01.023. PMID:16507361. Sugasawa, K., Masutani, C., and Hanaoka, F. 1993. Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. I. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes. J. Biol. Chem. 268: 9098–9104. PMID:8386176. Suquet, C., Mitchell, D.L., and Smerdon, M.J. 1995. Repair of UVinduced (6-4) photoproducts in nucleosome core DNA. J. Biol. Chem. 270: 507–509. doi:10.1074/jbc.270.2.507. PMID:7822270. Suter, B., and Thoma, F. 2002. DNA-repair by photolyase reveals dynamic properties of nucleosome positioning in vivo. J. Mol. Biol. 319: 395–406. doi:10.1016/S0022-2836(02)00291-7. PMID:12051916. Suter, B., Livingstone-Zatchej, M., and Thoma, F. 1997. Chromatin structure modulates DNA repair by photolyase in vivo. EMBO J. 16: 2150–2160. doi:10.1093/emboj/16.8.2150. PMID:9155040. Suter, B., Livingstone-Zatchej, M., and Thoma, F. 1999. Mapping cyclobutane-pyrimidine dimers in DNA and using DNA-repair by photolyase for chromatin analysis in yeast. Methods Enzymol. 304: 447–461. doi:10.1016/S0076-6879(99)04027-6. PMID:10372376. Suter, B., Wellinger, R.E., and Thoma, F. 2000a. DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair. Nucleic Acids Res. 28: 2060–2068. doi:10.1093/nar/28.10.2060. PMID:10773073. Suter, B., Schnappauf, G., and Thoma, F. 2000b. Poly(dA.dT) sequences exist as rigid DNA structures in nucleosome-free yeast promoters in vivo. Nucleic Acids Res. 28: 4083–4089. doi:10. 1093/nar/28.21.4083. PMID:11058103. Teng, Y., Yu, Y., and Waters, R. 2002. The Saccharomyces cerevisiae histone acetyltransferase Gcn5 has a role in the photoreactivation and nucleotide excision repair of UV-induces cyclobutane pyrimidine dimmers in the MFA2 gene. J. Mol. Biol. 316: 489– 499. doi:10.1006/jmbi.2001.5383. PMID:11866513. Teng, Y., Yu, Y., Ferreiro, J.A., and Waters, R. 2005. Histone acetylation, chromatin remodelling, transcription and nucleotide excision repair in S. cerevisiae: studies with two model genes. DNA Repair (Amst.), 4: 870–883. doi:10.1016/j.dnarep.2005.04. 006. PMID:15950549. Thoma, F. 1999. Light and dark in chromatin repair: repair of UVinduced DNA lesions by photolyase and nucleotide excision repair. EMBO J. 18: 6585–6598. doi:10.1093/emboj/18.23.6585. PMID:10581233. Thoma, F. 2005. Repair of UV lesions in nucleosomes–intrinsic properties and remodeling. DNA Repair (Amst.), 4: 855–869. doi:10.1016/j.dnarep.2005.04.005. PMID:15925550. Tijsterman, M., De Pril, R., Tasseron-De Jong, J.G., and Brouwer, J. 1999. RNA polymerase II transcription suppresses nucleosomal modulation of UV-induced (6-4) photoproduct and cyclobutane pyrimidine dimer repair in yeast. Mol. Cell. Biol. 19: 934– 940. PMID:9858617.

Biochem. Cell Biol. Vol. 87, 2009 Tornaletti, S., and Hanawalt, P.C. 1999. Effect of DNA lesions on transcription elongation. Biochimie, 81(1–2): 139–146. doi:10. 1016/S0300-9084(99)80046-7. PMID:10214918. Tremblay, M., Teng, Y., Paquette, M., Waters, R., and Conconi, A. 2008. Complementary roles of yeast Rad4p and Rad34p in nucleotide excision repair of active and inactive rRNA gene chromatin. Mol. Cell. Biol. 28: 7504–7513. Toussaint, M., Levasseur, G., Tremblay, M., Paquette, M., and Conconi, A. 2005. Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I–transcribed ribosomal genes. Biochem. Cell Biol. 83: 449–459. doi:10.1139/ o05-141. PMID:16094448. Ura, K., and Hayes, J.J. 2002. Nucleotide excision repair and chromatin remodeling. Eur. J. Biochem. 269: 2288–2293. doi:10. 1046/j.1432-1033.2002.02888.x. PMID:11985610. Ura, K., Araki, M., Saeki, H., Masutani, C., Ito, T., Iwai, S., et al. 2001. ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes. EMBO J. 20: 2004–2014. doi:10.1093/emboj/20.8. 2004. PMID:11296233. van Noort, J., Orsini, F., Eker, A., Wyman, C., de Grooth, B., and Greve, J. 1999. DNA bending by photolyase in specific and nonspecific complexes studied by atomic force microscopy. Nucleic Acids Res. 27: 3875–3880. doi:10.1093/nar/27.19.3875. PMID:10481027. Verhage, R.A., Van De Putte, P., and Brouwer, J. 1996. Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strandspecific nucleotide excision repair of RNA polymeras I transcribed genes. Nucleic Acids Res. 24: 1020–1025. doi:10.1093/ nar/24.6.1020. PMID:8604332. Verhage, R.A., Tijsterman, M., van de Putte, P., and Brouwer, J. 1998. Transcription-coupled and global genome nucleotide excision repair. In Nucleic acids and molecular biology. Vol. 12. Edited by F. Eckstein, D.M.J. Lilley. Springer–Verlag, New York. pp. 157–172. Verreault, A. 2000. De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev. 14: 1430–1438. PMID:10859162. Wang, Z.G., Wu, X.H., and Friedberg, E.C. 1991. Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes. J. Biol. Chem. 266: 22472–22478. PMID:1939267. Waters, R., and Smerdon, M.J. 2005. Nucleotide excision repair in chromatin: searching for the key to enter. DNA Repair (Amst.), 4(Suppl): 853–950. doi:10.1016/j.dnarep.2005.04.004. PMID:15967734. Weber, S. 2005. Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase. Biochim. Biophys. Acta, 1707: 1–23. doi:10.1016/j.bbabio.2004.02.010. PMID:15721603. Wellinger, R.E., and Thoma, F. 1997. Nucleosome structure and positioning modulate nucleotide excision repair in the nontranscribed strand of an active gene. EMBO J. 16: 5046– 5056. doi:10.1093/emboj/16.16.5046. PMID:9305646. Yu, Y., Teng, Y., Liu, H., Reed, S.H., and Waters, R. 2005. UV irradiation stimulates histone acetylation and chromatin remodeling at a repressed yeast locus. Proc. Natl. Acad. Sci. U.S.A. 102: 8650–8655. doi:10.1073/pnas.0501458102. PMID:15939881.

Published by NRC Research Press