Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability.
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.
Damerla
Rama Rao
RR
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, PA 15219, United States.
Knickelbein
Kelly E
KE
Kepchia
Devin
D
Jackson
Abbe
A
Armitage
Bruce A
BA
Eckert
Kristin A
KA
Opresko
Patricia L
PL
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D053960:DNA Breaks; D004261:DNA Replication / Q000235:genetics; D043542:DNA, Z-Form / Q000737:chemistry; D054856:G-Quadruplexes; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D016813:Oxytricha / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D016615:Telomere / Q000235:genetics*; D013696:Temperature; D016808:Tetrahymena thermophila / Q000235:genetics
D043542:DNA, Z-Form; C051802:triplex DNA; D004247:DNA
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