Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light

Assendelft, Greet Blom van; Rigney, Elizabeth; Hickson, Ian

doi:10.1093/nar/21.15.3399

Cited by 23 publications

(19 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Preparation of Oligonucleotides, Irradiation with UV Light-The oligonucleotides used in this study were designed as described previously (29) and synthesized on a Gene Assembler Plus DNA Synthesizer (Amersham Pharmacia Biotech) using the phosphoramidite method. The adjacent pyrimidines are underlined (see below).…”

Section: Methodsmentioning

confidence: 99%

Preferential Binding of High Mobility Group 1 Protein to UV-damaged DNA

Pasheva¹,

Pashev²,

Favre³

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 ؎ 0.5 and 4.0 ؎ 1.5 M for irradiated TT and TC oligonucleotides, respectively, and 70 ؎ 20 M for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed.High mobility group proteins 1 and 2 (HMG1 and -2) 1 are abundant chromosomal proteins found in a variety of eukaryotic species. Despite their early identification and biochemical characterization (1) and subsequent implication in a number of cellular events, their functions are still unknown (for reviews see Refs. 2 and 3). The interest in these proteins sharply increased in the early 1990s with the discovery of a new kind of eukaryotic protein domain involved in interactions with DNA (4), which displayed sequence similarity to two homologous repeats of an 80-amino acid sequence in HMG1. This motif called HMG box was found in a wealth of less abundant eukaryotic proteins, mainly general transcription factors and gene-specific transcriptional activators (reviewed in Refs. 3 and 5). A general characteristic of these proteins is their ability to bind bent DNA or to induce bending in DNA independent of sequence or the existence of prebent regions (6). Thus, HMG1 binds preferentially to distorted DNA sites such as synthetic cruciform DNA structures (7,8) and the lesions formed on DNA by the anticancer drug cisplatin (9, 10). The ability of HMG box-containing proteins to mediate bending in linear duplex DNA was demonstrated by several approaches (11-13). In addition, these proteins can constrain negative supercoils in plasmid DNA (14 -16) and can serve as "architectural" elements in the assembly of higher order nucleoprotein complexes (5,11,13,(17)(18)(19).The finding that HMG1 binds more tightly to cisplatin-damaged DNA than to unmodified B form DNA implies that the signal f...

show abstract

Section: Methodsmentioning

confidence: 99%

Preferential Binding of High Mobility Group 1 Protein to UV-damaged DNA

Pasheva¹,

Pashev²,

Favre³

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…DDB was originally identified as an activity that binds to UV-damaged DNA (3). It has high affinities for the 6-4 photoproduct (14,29,36). In addition, DDB also binds to cisplatin-modified DNA (14).…”

mentioning

confidence: 99%

“…In addition, DDB also binds to cisplatin-modified DNA (14). It has been proposed that the damaged-DNA binding activity of DDB is related to a potential DNA repair function (7,15,18,20,36). The microinjection of purified DDB overcomes the repair deficiencies in cells from XP-E patients lacking the damaged-DNA binding activity of DDB (20, 27).…”

mentioning

confidence: 99%

The Naturally Occurring Mutants of DDB Are Impaired in Stimulating Nuclear Import of the p125 Subunit and E2F1-Activated Transcription

Shiyanov

Hayes

Donepudi

et al. 1999

Molecular and Cellular Biology

101

View full text Add to dashboard Cite

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.The human UV-damaged-DNA binding protein has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E). Cells from about 30% of XP-E patients (6 of 19) were shown to lack the damaged-DNA binding activity of DDB (3,27). DDB was originally identified as an activity that binds to UV-damaged DNA (3). It has high affinities for the 6-4 photoproduct (14,29,36). In addition, DDB also binds to cisplatin-modified DNA (14). It has been proposed that the damaged-DNA binding activity of DDB is related to a potential DNA repair function (7,15,18,20,36). The microinjection of purified DDB overcomes the repair deficiencies in cells from XP-E patients lacking the damaged-DNA binding activity of DDB (20, 27). However, purified DDB has no significant effect in nucleotide excision repair assays in vitro (18). A recent study proposed that DDB functions as a repair protein in the context of chromatin structure (27). A repair function of DDB would be consistent with the fact that the p48 subunit of DDB possesses extensive sequence homology with the Cockayne syndrome protein CS-A, which is involved in transcription-coupled DNA repair (2,9,12,19).DDB also possesses a transcriptional functi...

show abstract

“…DDB was originally identified as a cellular activity that binds UVdamaged DNA (7,27,33,50,61). DDB activity was also found to be missing in cells from a subset (20%) of xeroderma pigmentosum complementation group E (XP-E) patients (7).…”

mentioning

confidence: 99%

DDB, a Putative DNA Repair Protein, Can Function as a Transcriptional Partner of E2F1

Hayes

Shiyanov

Chen

et al. 1998

Molecular and Cellular Biology

View full text Add to dashboard Cite

The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, we looked for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. Here we show that DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. We show that the UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. Moreover, the 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1 depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts resulted in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also revealed a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, was enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. We also observed evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulated E2F1-activated transcription. In addition, the transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 was stimulated by coexpression of DDB. Moreover, DDB expression could overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. We speculate that the damaged-DNA binding function of DDB, besides repair, might serve as a negative regulator of E2F1-activated transcription, as damaged DNA will sequester DDB and make it unavailable for E2F1. Furthermore, the binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA.E2F1 is the most-studied member of the E2F family of transcription factors. E2F1 binds the consensus E2F site (TT TCGCGC) as a heterodimer in conjunction with DP1, and it stimulates transcription both in vitro and in vivo (3,13,(20)(21)(22)31). Several genes that are essential for DNA replication and S-phase entry have been shown to be transcriptionally activated by overexpression of E2F1. Included are genes expressing dihydrofolate reductase (4, 26, 56), ribonucleotide reductase, PCNA, DNA polymerase ␣, thymidine kinase, cyclin E, cyclin A, and E2F1, as well as cdc2 (8,42,44). This is consistent with the observation that overexpression of E2F1 induces quiescent cells to enter S phase (2, 30).The activity and levels of E2F1 are regulated very tightly during the progression of the cell cycle. E...

show abstract

Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light

Cited by 23 publications

References 40 publications

Preferential Binding of High Mobility Group 1 Protein to UV-damaged DNA

Preferential Binding of High Mobility Group 1 Protein to UV-damaged DNA

The Naturally Occurring Mutants of DDB Are Impaired in Stimulating Nuclear Import of the p125 Subunit and E2F1-Activated Transcription

DDB, a Putative DNA Repair Protein, Can Function as a Transcriptional Partner of E2F1

Contact Info

Product

Resources

About