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The p53 protein shows exonuclease activity and degrades DNA with a 3'-to-5' polarity in the presence of Mg2+. The exonuclease activity is specific for the purified wild-type protein as p53 mutants lack the activity. The activity copurifies with p53 during the preparation of the wild-type protein but fails to copurify with mutant p53. The enzyme activity can be reconstituted from urea-denatured and SDS gel-purified p53 and mapped to the core region of the p53 molecule.

Abstract: Human DHX9 helicase, also known as nuclear DNA helicase II (NDH II) and RNA helicase A (RHA), belongs to the SF2 superfamily of nucleic acid unwinding enzymes. DHX9 melts simple DNA–DNA, RNA–RNA, and DNA–RNA strands with a 3′–5′ polarity; despite this little is known about its substrate specificity. Here, we used partial duplex DNA consisting of M13mp18 DNA and oligonucleotide-based replication and recombination intermediates. We show that DHX9 unwinds DNA- and RNA-containing forks, DNA- and RNA-containing displacement loops (D- and R-loops), and also G-quadruplexes. With these substrates, DHX9 behaved similarly as the RecQ helicase WRN. In contrast to WRN, DHX9 melted RNA-hybrids considerably faster than the corresponding DNA–DNA strands. DHX9 preferably unwound R-loops and DNA-based G-quadruplexes indicating that these structures may be biologically relevant. DHX9 also unwound RNA-based G-quadruplexes that have been reported to occur in human transcripts. It is believed that an improper dissolution of co-transcriptionally formed D-loops, R-loops, and DNA- or RNA-based G-quadruplexes represent potential roadblocks for transcription and thereby enhance transcription associated recombination events. By unwinding these structures, DHX9 may significantly contribute to transcriptional activation and also to the maintenance of genomic stability. [Copyright &y& Elsevier]

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, is involved in transcription and RNA processing. Here, we report that NDH II interacts with the Werner syndrome helicase WRN, an enzyme associated with premature aging and predisposition to tumorigenesis. NDH II was co-purified with WRN, DNA polymerase δ, and replication protein A (70 kDa) during several steps of conventional column chromatography. Co-immunoprecipitations revealed an association between NDH II, WRN, and polymerase δ. We demonstrate a direct protein-protein interaction between WRN and NDH II that is mediated by the N-terminal double-strand RNA-binding domain II and C-terminal RGG box of NDH II and the N-terminal exonuclease domain of WRN. WRN inhibited the DNA-dependent NTPase and DNA helicase activities of NDH II. On the other hand, the 3′ ↵ 5′ exonuclease activity of WRN was increased by the presence of NDH II. NDH II directly stimulated the exonuclease domain of WRN, whereas the exonuclease domain of WRN suppressed the DNA-dependent (but not RNA. dependent) ATPase activity of NDH II. These results suggest that the double-strand RNA-binding domain II and RGG box of NDH II together form a protein-protein interaction surface that contacts the exonuclease domain of WRN. Furthermore, NDH II enhanced the degradation of D-loop DNA by the WRN exonuclease. Taken together, these results suggest that NDH II plays a role in promoting the DNA processing function of WRN, which in turn might be necessary for maintaining genomic stability. [ABSTRACT FROM AUTHOR]

Formation of γ-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that γ-H2AX foci were also formed when cells were incubated with 0.5 µg/ml DNA intercalating agent actinomycin D. In untreated cells, γ-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with γ-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and γ-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to γ-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with γ-H2AX foci. Taken together, these results suggest that histone γ-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA. [ABSTRACT FROM AUTHOR]

DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest. This is accompanied by morphological defects, elevation of senescence-associated β-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression. [ABSTRACT FROM AUTHOR]

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif. [ABSTRACT FROM AUTHOR]

Simian virus 40 large tumor antigen (Tag) is a multifunctional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The Nterminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed. [ABSTRACT FROM AUTHOR]

Investigates the conformational changes, which accompany the binding of human replication protein A (RPA) to partial DNA duplexes and assigned protein domains. Interaction of RPA with various ssDNA substrates; Different DNA structures partial DNA duplexes; Proteolytic profile of RPA digested to trypsin.