TY - JOUR
T1 - Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability
AU - Damerla, Rama Rao
AU - Knickelbein, Kelly E.
AU - Kepchia, Devin
AU - Jackson, Abbe
AU - Armitage, Bruce A.
AU - Eckert, Kristin A.
AU - Opresko, Patricia L.
N1 - Funding Information:
This work was supported by NIH grants [ RO1 ES0515052 (P.L.O.); RO1 CA100060 (K.A.E.)], the Ellison Medical Foundation (P.L.O.), and the Jake Gittlen Cancer Research Foundation (K.A.E.). We thank members of the Opresko and Eckert lab for critical reading of the manuscript, and Gregory Sowd and Suzanne Hile for technical support and assistance.
PY - 2010/11/10
Y1 - 2010/11/10
N2 - Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]10 repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]4 and [TTTTGGGG]4 are thermally more stable compared to human [TTAGGG]4. Consistent with this, replication of vectors with ciliate [TTGGGG]10 repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]10 vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.
AB - Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]10 repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]4 and [TTTTGGGG]4 are thermally more stable compared to human [TTAGGG]4. Consistent with this, replication of vectors with ciliate [TTGGGG]10 repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]10 vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.
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U2 - 10.1016/j.dnarep.2010.07.014
DO - 10.1016/j.dnarep.2010.07.014
M3 - Article
C2 - 20800555
AN - SCOPUS:78049373384
VL - 9
SP - 1119
EP - 1129
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
IS - 11
ER -