RESUMO
The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C-containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C->T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Histona-Lisina N-Metiltransferase , Dímeros de Pirimidina , Humanos , Camundongos , DNA/metabolismo , DNA/química , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Histonas/genética , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
To recognize DNA adducts, nucleotide excision repair (NER) deploys the XPC sensor, which detects damage-induced helical distortions, followed by engagement of TFIIH for lesion verification. Accessory players ensure that this factor handover takes place in chromatin where DNA is tightly wrapped around histones. Here, we describe how the histone methyltransferase ASH1L, once activated by MRG15, helps XPC and TFIIH to navigate through chromatin and induce global-genome NER hotspots. Upon UV irradiation, ASH1L adds H3K4me3 all over the genome (except in active gene promoters), thus priming chromatin for XPC relocations from native to damaged DNA. The ASH1L-MRG15 complex further recruits the histone chaperone FACT to DNA lesions. In the absence of ASH1L, MRG15 or FACT, XPC is misplaced and persists on damaged DNA without being able to deliver the lesions to TFIIH. We conclude that ASH1L-MRG15 makes damage verifiable by the NER machinery through the sequential deposition of H3K4me3 and FACT.
Assuntos
Cromatina , Histonas , Histonas/genética , Reparo do DNA , MetiltransferasesRESUMO
The potential of urate oxidase (uricase) for clinical use has been highlighted because of its role in lowering the blood uric acid levels for the treatment of tumor lysis syndrome. In the present study, the codon-optimized synthetic gene of Aspergillus flavus uricase was fused to the Mxe GyrA intein and chitin-binding domain. The construct was inserted into pPICZA and pPICZαA vectors and electroporated into Pichia pastoris GS115 for the cytosolic and secretory expression. Transformants were screened on gradients of Zeocin up to 2000 µg/ml to find multi-copy integrants. For both constructs, colonies with more resistance were screened for the highest uricase producers by enzyme assay. PCR analysis confirmed successful cassettes insertion into the genome and Mut + phenotype. The gene copy index was determined to be two and five for cytosolic and secretory strains, respectively. Productivity of the cytosolic and secretory strains was found to be 0.74 and 0.001 U/ml culture media in order while the cytosolic recombinant enzyme accounted for about 6% of total proteins. One-step purification of the expressed uricase was done with the aid of the chitin affinity column, followed by DTT induction for intein on-column cleavage. The yield of 40.8 mg/L and K m of 0.22 mM was obtained for intracellular expression. It seems that the intracellular production of uricase can indeed serve as an effective alternative to secretory expression. Moreover, this is the first report considering cytosolic production of uricase using the intein-mediated protein purification in the methylotrophic yeast, P. pastoris.