RESUMEN
L1 retrotransposons comprise 17% of the human genome and are its only autonomous mobile elements. Although L1-induced insertional mutagenesis causes Mendelian disease, their mutagenic load in cancer has been elusive. Using L1-targeted resequencing of 16 colorectal tumor and matched normal DNAs, we found that certain cancers were excessively mutagenized by human-specific L1s, while no verifiable insertions were present in normal tissues. We confirmed de novo L1 insertions in malignancy by both validating and sequencing 69/107 tumor-specific insertions and retrieving both 5' and 3' junctions for 35. In contrast to germline polymorphic L1s, all insertions were severely 5' truncated. Validated insertion numbers varied from up to 17 in some tumors to none in three others, and correlated with the age of the patients. Numerous genes with a role in tumorigenesis were targeted, including ODZ3, ROBO2, PTPRM, PCM1, and CDH11. Thus, somatic retrotransposition may play an etiologic role in colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/genética , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Metilación , Inestabilidad de Microsatélites , Mutagénesis Insercional , Fenotipo , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
L1-seq is a high-throughput sequencing technique which is utilized to identify novel L1 insertions in genomic DNA samples of interest. Using special diagnostic nucleotides unique to the youngest and most active L1 sequence, we can amplify new somatic insertions. This technique has helped to establish the number of L1 insertions present in the general population as well as the variation among individuals with regard to their complement of active L1 elements. More recently, this technique has been employed to assess the level of retrotransposition occurring in various diseases such as cancer. These efforts try to establish a connection between the process of retrotransposition and disease development and/or progression.
Asunto(s)
Genoma Humano , Genómica , Elementos de Nucleótido Esparcido Largo , Biología Computacional/métodos , Biblioteca Genómica , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reproducibilidad de los ResultadosRESUMEN
Acetate kinase (ACK), which catalyzes the reversible phosphorylation of acetate by ATP, is a member of the acetate and sugar kinase/heat shock cognate/actin (ASKHA) superfamily. ASKHA family members share a common core fold that includes an ATPase domain with five structural motifs. The PHOSPHATE1 motif has previously been shown to be important for catalysis. We have investigated the role of two of these motifs in the Methanosarcina thermophila ACK (MtACK) and have shown that residues projecting into the ACK active site from the PHOSPHATE2 and ADENOSINE loops and a third highly conserved loop designated here as LOOP3 play key roles in nucleotide triphosphate (NTP) selection and utilization. Alteration of Asn211 of PHOSPHATE2, Gly239 of LOOP3, and Gly331 of ADENOSINE greatly reduced catalysis. In particular, Gly331, which is highly conserved throughout the ASKHA superfamily, has the greatest effect on substrate selection. Alteration at this site strongly skewed MtACK toward utilization of purines over pyrimidines, unlike the wild type enzyme that shows broad NTP utilization. Further investigation into differences between the ATPase domain in MtACK and other acetate kinases that show different substrate preferences will provide us with a better understanding of the diversity of phosphoryl donor selection in this enzyme family.