The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity.
The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity.
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DOI：
10.1093/nar/gkm002
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发表时间：
2007
影响因子：
14.9
通讯作者：
Prasad VR
中科院分区：
文献类型：
--
作者：
Drosopoulos WC;Prasad VR
人端粒酶逆转录酶（HTERT）是人端粒酶的催化亚基，包含逆转录病毒逆转录酶和端粒酶的保守基序。 Tyr183和Met184的位置定向诱变保留在HIV-1 RT中，保留类似于Leu866和Val867，他们表明，它们是核OTIDE结合，加工性和保真度的关键决定者，我们显示Val867的替换会导致整体酶活性和端粒重复扩展率的显着变化，但对聚合酶加工的影响很小检查（ALA，MET，THR）导致重复延伸率降低，范围从野生型率的20％到50％。带有突变模板序列的端粒酶RNA（TRS）揭示了对延伸率的影响与模板复制缺陷有关，而对模板A残差也是如此。这些发现表明，通过在867的位置有一个阀聚合酶保真度以最佳和快速重复合成。
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from ∼20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis.