Improved method for measuring the ensemble average of strand breaks in genomic DNA

Bespalov, Vyacheslav A.; Conconi, Antonio; Zhang, Xiaoyi; Fahy, Deirdre; Smerdon, Michael J.

doi:10.1002/em.1068

Cited by 23 publications

(44 citation statements)

References 37 publications

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“…Radioactive probes were generated from EcoRI-digested MCF-7 cell genomic DNA followed by random priming (RadPrime™; Invitrogen). Quantification of CPDs was performed on data from phosphorimages of the Southern blots of these gels, using ImageQuant software and number-average DNA length analysis (39).…”

Section: Methodsmentioning

confidence: 99%

“…Random hybridization probes are then used to detect all genomic DNA sequences with minimal sequence bias. Analysis of scans of the phosphorimage signal profiles of the heterogeneous populations of DNA fragments on the membrane can be integrated to determine the number-average size of DNA fragments in each lane (39). As illustrated in Fig.…”

Section: Overexpression Of Hmga1 Proteins Inhibits Ner In Mcf7 Cells-mentioning

confidence: 99%

“…3A, genomic DNA isolated from cells immediately after UV irradiation (0 h repair) and digested with T4 endo V (ϩ lane) migrates as a smear of heterogeneous single-stranded fragments whose ensemble average size (indicated by an asterisk) can be accurately determined (Ref. 39 and references therein). This is in contrast to the DNA not treated with T4 endo V, which migrates only a short distance into the gel (Fig.…”

Section: Overexpression Of Hmga1 Proteins Inhibits Ner In Mcf7 Cells-mentioning

confidence: 99%

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Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

Adair¹,

Kwon²,

Dement

et al. 2005

Journal of Biological Chemistry

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The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-footprinting" demonstrated that CPD formation within a long run of Ts (T 18 -tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.In most organisms, DNA helix-distorting bulky lesions are repaired by nucleotide excision repair (NER) 5 involving the excision and replacement of 24 -32 nt of the damaged DNA strand (1). Many factors, such as (a) the type of DNA damage, (b) the DNA sequence surrounding the lesion, (c) the position in chromatin, and (d) the interactions with DNAbinding proteins, are known to affect the efficiency of NER (2-4). For instance, inhibitory effects of nucleosomes on NER have been observed in vitro and in intact cells (5-9), presumably reflecting the limited access of NER proteins to these lesions (10). In a similar way, certain DNAbinding proteins, such as transcription factor IIIA and HMGB1 proteins, are known to repress NER at their cognate sequences (11-13). Conversely, transcriptional activators and the RNA polymerase II elongation complex are associated with enhanced repair of transcribing genes (14, 15). Furthermore, in contrast to HMGB1, another member of the HMG protein superfamily that specifically binds to nucleosomes, HMGN1, has been shown to likewise enhance NER of UV-damaged DNA in vivo (16).Stable DNA photoproducts produced by UV-induced covalent linkage between adjacent bases are prototypes of helical distorting, bulky lesions (17). Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known...

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Section: Methodsmentioning

confidence: 99%

Section: Overexpression Of Hmga1 Proteins Inhibits Ner In Mcf7 Cells-mentioning

confidence: 99%

Section: Overexpression Of Hmga1 Proteins Inhibits Ner In Mcf7 Cells-mentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

Adair¹,

Kwon²,

Dement

et al. 2005

Journal of Biological Chemistry

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“…The number of CPDs present in genomic DNA was determined as described (35). DNA samples (5-10 g) were digested with T4 endo V and resolved on 1% alkaline agarose gels.…”

Section: Methodsmentioning

confidence: 99%

Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

Conconi

Bespalov

Smerdon

2002

Proc. Natl. Acad. Sci. U.S.A.

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Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. N ucleotide excision repair (NER) removes different types of lesions from DNA, including bulky adducts caused by chemicals, interstrand or intrastrand crosslinks, and the UV photoproducts cis-syn cyclobutane pyrimidine dimer (CPD) and pyrimidine (6-4) pyrimidone (1). If DNA lesions are not removed, mutations can occur after translesion replication (2). It is now well established that many transcriptionally active genes are repaired faster than inactive DNA (3, 4). Furthermore, preferential removal of CPDs from active genes is caused mainly by an increased rate of repair of the transcribed strand (TS). This transcription-coupled repair (TCR) was first discovered in mammalian cells (5), then in Escherichia coli (6) and yeast (7). Elongation by RNA polymerase II (pol II) is required for TCR (8, 9), and it is thought that only pol II-transcribed genes are subject to TCR (10).RNA polymerase I (pol I) transcribes ribosomal genes (rDNA) at a very high rate. The rDNA is localized in the nucleolus, which is a dense chromatin region composed of rDNA, pol I, rRNA, assembling ribosomes, and proteins involved in cell-cycle regulation (11,12). It is known that mammalian cells repair rDNA damaged by UV radiation (13) and chemicals (14-16). However, in rodent and human cells, CPDs are less efficiently repaired in rDNA than in either total genomic DNA or pol II-transcribed genes (16)(17)(18)(19)(20). Moreover, DNA repair does not exhibit strand bias in the rDNA of these cells (17,19). To the contrary, CPDs are rapidly removed from both strands of total rDNA in yeast (21,22). In addition, a strand bias during repair of total rDNA was observed in rad7 and rad16 mutants, even though strand-specific repair was not observed in total rDNA of wild-type cells (21).Ribosomal genes are present in multiple copies organized in long tandem repeats (11), and in most cells only a f...

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“…Afterward, single-strand DNA was resolved on a 1.2% alkaline gel and stained with SYBR Gold (Invitrogen). The gel was scanned by Typhoon FLA 7000 (GE Healthcare), and quantifications were performed using ImageQuant 5.2 (Molecular Dynamics) to determine the number of CPDs per kilobase as previously described (Bespalov et al 2001). …”

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confidence: 99%

Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability inSaccharomyces cerevisiae

Hodges

Gallegos

Laughery³

et al. 2015

Genetics

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A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue.KEYWORDS nucleotide excision repair; cryptic transcription; nucleosome; histone assembly T HE histone octamer, which is composed of two copies each of histones H2A, H2B, H3, and H4, binds with high affinity to $147 bp of DNA to form the nucleosome core particle. Histone-DNA binding is mediated by .100 histone main-chain and side-chain interactions with the DNA sugarphosphate backbone and a similar number of indirect water-mediated interactions (Davey et al. 2002;Muthurajan et al. 2003). These interactions occur primarily at 14 locations in the nucleosome structure where the DNA minor groove faces the histone octamer [superhelical locations (SHL) 26.5 to 6.5] (Luger et al. 1997). At each of these locations, an arginine side chain extends into the DNA minor groove and makes extensive contacts with the DNA backbone ( Figure 1A).We will refer to the arginine residues that insert into the DNA minor groove as "sprocket" arginines, since they engage the DNA "chain" like the teeth of a bicycle sprocket wheel. Sprocket arginine residues are highly conserved (Muthurajan et al. 2003;Sullivan and Landsman 2003), and the insertion of sprocket arginine side chains into the DNA minor groove comprises a significant fraction of the solvent accessible surface area that is buried upon histone-DNA binding (Davey et al. 2002). Studies have suggested that sprocket arginine-DNA contacts may play an important role in the rotational positioning of nucleosomes (Luger et al. 1997;Harp et al. 2000;Rohs et al. 2009;Wang et al. 2010;West et al. 2010). For example, short poly(A) stretches narrow the DNA minor groove, potentially enhancing electrostatic interactions between the DNA phosphate backbone and the sprocket arginine (Rohs et al. 2009;West et al. 201...

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Improved method for measuring the ensemble average of strand breaks in genomic DNA

Cited by 23 publications

References 37 publications

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability inSaccharomyces cerevisiae

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