|Regulation of 5' template usage and incorporation of noncognate nucleotides by human telomerase |
Moriarty TJ, Marie-Egyptienne DT, Autexier C
Category: telomerase ¤ Added: Mar 17, 2006 ¤ Rating: ◊◊
Telomerase accurately synthesizes telomeric DNA by reverse transcription of a tightly defined template region in the telomerase RNA (TR). Reverse transcription past the 50 boundary of the template can cause the incorporation of noncognate nucleotides into telomeric DNA, which can result in disruption of normal telomere function. The products synthesized by human telomerase do not contain the nucleotide cytosine, which is encoded by an hTR residue 2 nucleotides (nt) 50 of the template boundary. We examined dCTP incorporation by a series of telomerases reconstituted with N- and C-terminally mutated human telomerase reverse transcriptases (hTERTs). We found that altering sequences in the N-terminal RNA interaction domain 1 (RID1) and C terminus caused dCTP-dependent catalytic phenotypes suggestive of reverse transcription of sequences 50 of the template boundary. A RID1 mutant that exhibited a dCTP-dependent phenotype interacted less efficiently with a human telomerase RNA (hTR) variant in which the 50 template boundary-defining P1b element was disrupted, whereas C-terminal mutations did not alter hTR interactions in a P1b-dependent fashion. Disruption of P1b or template linker sequences between P1b and the 50 template boundary also impaired 50 template usage in RID1 and C-terminal hTERT mutants. These observations identify overlapping roles for hTR sequences and structures 50 of the template in regulating both 50 template boundary definition and 50 template usage, and implicate hTERT N- and C-terminal regions in 50 template usage and suppression of noncognate nucleotide incorporation.