Enhanced DNA repair synthesis in hyperacetylated nucleosomes

We have investigated the level of "early" DNA repair synthesis in nucleosome subpopulations, varying in histone acetylation, from normal human fibroblasts treated with sodium butyrate. We find that repair synthesis occurring during the first 30 min after UV irradiation is significantly enhanced in hyperacetylated mononucleosomes. Nucleosomes with an average of 2.3 acetyl residues/H4 molecule contained approximately 1.8-fold more repair synthesis than nucleosomes with an average of 1.5 or 1.0 acetyl residues/H4 molecule. Fractionation of highly acetylated nucleosomes by two-dimensional gel electrophoresis yielded an additional 2.0-fold enrichment of repair synthesis in nucleosomes containing 2.7 acetyl residues/H4 molecule as compared to nucleosomes containing 1.9 acetyl residues/H4 molecule. This enhanced repair synthesis is associated primarily with nucleosome core regions and does not appear to result from increased UV damage in hyperacetylated chromatin. In addition, the distribution of repair synthesis within nucleosome core DNA from hyperacetylated chromatin is nonrandom, showing a bias toward the 5' end which is similar to that obtained for bulk (unfractionated) chromatin. These results provide strong evidence that enhanced repair occurs within nucleosome cores of hyperacetylated chromatin in butyrate-treated human cells. Finally, pulse-chase experiments demonstrate that the association of enhanced repair synthesis with hyperacetylated nucleosomes is transient, lasting only about 12 h after UV damage.     

Deletion analysis of a unique 3'' splice site indicates that alternating guanine and thymine residues represent an efficient splicing signal.

The 3' splice site of the second intron (I2) of the human apolipoprotein-AII gene, (GT)16GGGCAG, is unique in that, although fully functional, a stretch of alternating guanine and thymine residues replaces the polypyrimidine tract usually associated with 3' splice junctions. The transient expression of successive 5' deletion mutants has defined the minimum number of nucleotides at the 3' end of apo-AII I2 that are required to direct efficient splicing. Processing in two cell-types, representing apo-AII producing and non-producing tissue was identical; in both, only by removing all the GT repeats did the 3' splice site of apo-AII I2 become completely non-functional. Similar deletion analyses of "classic" 3' splice sites, which conform to the consensus sequence (Y)nNYAG, have indicated that a minimum of 14 nucleotides of the polypyrimidine tract are required for detectable levels of processing to take place. Here we report that the six nucleotides (GT)2GG, which directly replace this tract in a deletion mutant of the 3' splice site of apo-AII I2 are sufficient to direct the splicing process efficiently and correctly.     

Protein splicing mechanisms and applications

Inteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches.     

Two DNA-binding sites on the globular domain of histone H5 are required for binding to both bulk and 5 S reconstituted nucleosomes

We have previously shown the existence of two DNA-binding sites on the globular domain of H5 (termed GH5), both of which are required for nucleosome organisation, as judged by the protection of a 166 bp chromatosome intermediate during micrococcal nuclease digestion of chromatin. This supports a model in which GH5 contacts two duplexes on the nucleosome. However, studies of a nucleosome assembled on the 5 S rRNA gene have argued against the requirement for two DNA-binding sites for chromatosome protection, which has implications for the role of linker histones. We have used this proposed difference in the requirement for a second site on the globular domain in the two models as a means of investigating whether bulk and reconstituted 5 S nucleosomes are indeed fundamentally different. GH5 protects a 166 bp chromatosome in both "bulk" and 5 S systems, and in both cases protection is abolished when all four basic residues in site II are replaced by alanine. Binding to four-way DNA junctions, which present a pair of juxtaposed duplexes, is also abolished. Single mutations of the basic residues did not abolish chromatosome protection in either system, or binding to four-way junctions, suggesting that the residues function as a cluster. Both bulk and 5 S nucleosomes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode of binding in each case, in which two DNA duplexes are contacted in the nucleosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes.     

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.     

Photosensitized DNA damage induced by NADH: site specificity and mechanism

Increasing evidence reveals the carcinogenicity of UVA radiation. We demonstrated that UVA-irradiated NADH induced damage to (32)P-labeled DNA fragments obtained from the p53 gene in the presence of Cu(II). Formamidopyrimidine glycosylase (Fpg)-sensitive lesions were formed at guanine residues, whereas piperidine-labile lesions occurred frequently at thymine residues. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), upon UVA exposure in the presence of Cu(II), increased depending on NADH concentration. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of reactive species derived from H(2)O(2) and Cu(I). UVA-irradiated riboflavin induced DNA cleavage through electron transfer at 5' guanine of the 5'-GG-3' sequence with both Fpg and piperidine treatments; Fpg induced less cleavage at the guanine residues than piperidine. These results imply that NADH may participate as an endogenous photosensitizer in UVA carcinogenesis via H(2)O(2) generation, producing metal-mediated mutagenic lesions such as 8-oxodG.     

Reconstruction of ancestral protosplice sites

Most of the eukaryotic protein-coding genes are interrupted by multiple introns. A substantial fraction of introns occupy the same position in orthologous genes from distant eukaryotes, such as plants and animals, and consequently are inferred to have been inherited from the common ancestor of these organisms. In contrast to these conserved introns, many other introns appear to have been gained during evolution of each major eukaryotic lineage. The mechanism(s) of insertion of new introns into genes remains unknown. Because the nucleotides that flank splice junctions are nonrandom, it has been proposed that introns are preferentially inserted into specific target sequences termed protosplice sites. However, it remains unclear whether the consensus nucleotides flanking the splice junctions are remnants of the original protosplice sites or if they evolved convergently after intron insertion. Here, we directly address the existence of protosplice sites by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and accordingly are not subject to selection for splicing efficiency. We show that introns are either predominantly inserted into specific protosplice sites, which have the consensus sequence (A/C)AG/Gt, or that they are inserted randomly but are preferentially fixed at such sites.     

Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase

How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.     

Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends

Nonhomologous end joining (NHEJ) is essential for efficient repair of chromosome breaks. However, the NHEJ ligation step is often obstructed by break-associated nucleotide damage, including base loss (abasic site or 5'-dRP/AP sites). Ku, a 5'-dRP/AP lyase, can excise such damage at ends in preparation for the ligation step. We show here that this activity is greatest if the abasic site is within a short 5' overhang, when this activity is necessary and sufficient to prepare such termini for ligation. In contrast, Ku is less active near 3' strand termini, where excision would leave a ligation-blocking α,β-unsaturated aldehyde. The Ku AP lyase activity is also strongly suppressed by as little as two paired bases 5' of the abasic site. Importantly, in vitro end joining experiments show that abasic sites significantly embedded in double-stranded DNA do not block the NHEJ ligation step. Suppression of the excision activity of Ku in this context therefore is not essential for ligation and further helps NHEJ retain terminal sequence in junctions. We show that the DNA between the 5' terminus and the abasic site can also be retained in junctions formed by cellular NHEJ, indicating that these sites are at least partly resistant to other abasic site-cleaving activities as well. High levels of the 5'-dRP/AP lyase activity of Ku are thus restricted to substrates where excision of an abasic site is required for ligation, a degree of specificity that promotes more accurate joining.     

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Site-specific oxidation at GG and GGG sequences in double-stranded DNA by benzoyl peroxide as a tumor promoter

Benzoyl peroxide (BzPO), a free-radical generator, has tumor-promoting activity. As a method for approaching the mechanism of tumor promoter function, the ability of oxidative DNA damage by BzPO was investigated by using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. BzPO induced piperidine-labile sites at the 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of Cu(I), whereas the damage occurred at single guanine residues of single-stranded DNA. Both methional and dimethyl sulfoxide (DMSO) inhibited DNA damage induced by BzPO and Cu(I), but typical hydroxyl radical ((*)OH) scavengers, superoxide dismutase (SOD) and catalase, did not inhibit it. On the other hand, H(2)O(2) induced piperidine-labile sites at cytosine and thymine residues of double-stranded DNA in the presence of Cu(I). Phenylhydrazine, which is known to produce phenyl radicals, induced Cu(I)-dependent damage at thymine residues but not at guanine residues. These results suggest that the BzPO-derived reactive species causing DNA damage is different from (*)OH and phenyl radicals generated from benzoyloxyl radicals. BzPO/Cu(I) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in double-stranded DNA more effectively than that in single-stranded DNA. Furthermore, we observed that BzPO increased the amount of 8-oxodG in human cultured cells. Consequently, it is concluded that benzoyloxyl radicals generated by the reaction of BzPO with Cu(I) may oxidize the 5'-guanine of GG and GGG sequences in double-stranded DNA to lead to 8-oxodG formation and piperidine-labile guanine lesions, and the damage seems to be relevant to the tumor-promoting activity of BzPO.     

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the α-helices and Β-strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the α-helix and the (Β-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.     

Identification of regions of potential flexibility in protein structures: folding units and correlations with intron positions

An algorithm has been developed to estimate flexibility for potential hinge motion at specified residues, that is, the mutual movement of two domains by rotation around a set of main-chain dihedral angles with torsion angles of neighboring side chains as variables. Such conformational changes must occur without severe atomic collisions. Flexible hinges have been found that satisfy such criteria. Sequence flexibility charts were obtained by plotting the flexibility of each residue against the residue number. Such charts were calculated for 10 proteins (ovomucoid third domain, cytochrome c, lysozyme, hemoglobin β-chain, α-chymotrypsin, elastase, carboxypeptidase A, dihydrofolate reductase, triosephosphate isomerase, and alcohol dehydrogenase) taken from the Protein Data Bank. The first step of unfolding is likely to occur at the hinge point with the largest flexibility. Following this idea, the polypeptide chain can be dissected into several folding units according to the sequence flexibility chart. When two domains are separated by conformational changes at such a hinge, the sequence flexibility chart for each domain changes, and it is recalculated and used to indicate subsequent unfolding steps. In this process of iterative estimation of flexibile hinges, some well-isolated hinges, or the border line between flexible and inflexible regions, were found to be directly at or close to the positions of splice junctions in the eukaryotic genes. Of a total of 45 splice junctions in the 10 proteins examined in this paper, 38 junctions can be identified as flexible hinges between folding units. We suggest that the iterative estimation of flexible hinges may define an array of possible folding/unfolding paths, and that the exon–intron arrangement in the gene may be closely correlated with the folding process of the protein.