In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere-binding protein TPP1 and the TEN domain of the telomerase catalytic subunit TERT regulate telomerase recruitment. TERT contains a unique domain, called the insertion in fingers domain (IFD), located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres, and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival. Telomeres are the protective nucleoprotein structures that cap the ends of linear eukaryotic chromosomes, thus preventing the aberrant and fatal activation of the DNA damage repair machinery. During normal somatic cell division, the end replication problem arising from the inability of DNA polymerase to completely replicate telomeres leads to progressive telomere loss and, over time, triggers cellular senescence to prevent carcinogenesis. The renewal capacity of germ cells, stem cells, and cancer cells is limited by telomere erosion and relies on the activation of a telomere maintenance mechanism for cellular survival. In over 85% of human cancers, detectable expression of telomerase, a specialized reverse transcriptase, is a requirement for cellular immortalization (1).In humans, telomerase is minimally composed of the core catalytic subunit human telomerase reverse transcriptase (hTERT) and an intrinsic RNA moiety, human telomerase RNA (hTR), to dictate the de novo synthesis of tandem TTAGGG repeats. Telomerase has the unique ability to synthesize long stretches of telomeric sequence repeats using its short RNA template through reiterative rounds of DNA synthesis, partial dissociation, translocation, and realignment with the newly synthesized telomere end. In human cells, this unique property, termed "repeat addition processivity" (RAP), is a determinant of telomere maintenance and cellular survival (2). The reverse transcriptase region of the TERT subunit contains seven motifs (1, 2, A, B=, C, D, and E) that are also conserved in other nucleic acid poly...

There is no evidence of measles virus persistence in the peripheral blood mononuclear cells of children with autism spectrum disorder.

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23 different integrase genes within the food-associated isolates, but only identified four different phages and integrase genes within clonal isolates of S. Enteritidis and S. Heidelberg. These results demonstrate the potential usefulness of PCR based detection of prophage integrase genes as a rapid indicator of genome diversity in S. enterica.

SummaryTelomerase is a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase (TERT), and an integrally associated RNA that contains a template for the synthesis of short repetitive G-rich DNA sequences at the ends of telomeres. Telomerase can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. The contribution of enzyme processivity to telomere length regulation in human cells is not well characterized. In cancer cells, under homeostatic telomere length-maintenance conditions, telomerase acts processively, whereas under nonequilibrium conditions, telomerase acts distributively on the shortest telomeres. To investigate the role of increased telomerase processivity on telomere length regulation in human cells with limited lifespan that are dependent on human TERT for lifespan extension and immortalization, we mutated the leucine at position 866 in the reverse transcriptase C motif of human TERT to a tyrosine (L866Y), which is the amino acid found at the equivalent position in HIV-1 reverse transcriptase. We report that, similar to the previously reported gain-of-function Tetrahymena telomerase mutant (L813Y), the human telomerase variant displays increased processivity. Human TERT-L866Y, like wild-type human TERT, can immortalize and extend the lifespan of limited-lifespan cells. Moreover, cells expressing human TERT-L866Y display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres, which is accompanied by telomere trimming events. Our results suggest that telomere length and homeostasis in human cells may be regulated by telomerase enzyme processivity.