The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy. DNA slippage during replicative synthesis appears to cause TRE. The causes of DNA slippage, however, remain mostly unknown. We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase. Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5 end of the template strand, induced dramatic TRE during DNA synthesis. The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract. Placing the abasic site in the primer did not induce TRE. TRE was sequence-dependent. The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT ϳ ATT > CAG > CTG. The expansions required replication from a primer complementary to the repeat tract. The expanded tracts were sequenced and contained multiple additions of the original repeat. The results imply that DNA damage can play a significant role in generating TRE in vivo.The occurrence of triplet-repeat expansion (TRE) 1 during transmission of genetic information is involved in many diseases including Fragile X syndrome (1-3), the most common form of mental retardation; myotonic dystrophy, a neuromuscular disorder; and several neurodegenerative disorders (1, 2). DNA slippage (4) during replicative synthesis is believed to be a major contributor to TRE in vivo (5-8), because elimination of recombination in yeast (9) and in Escherichia coli (10) does not influence TRE, whereas elimination of mismatch repair does (10, 11). Pausing or blockage of DNA replication has been proposed to promote slippage by giving the DNA replication complex more time to dissociate and form misaligned DNA intermediates (12, 13). Hairpins (14 -18), bulges (19,20), tetraplexes (18, 21), and possibly "slipped" structures (22, 23) have been proposed to promote DNA slippage. The structures were proposed to act either as intermediates, by their formation within the repeat or by blocking or pausing replication toward the end of the repeat (12, 24) or a combination of both (13,17,24). Replication pause sites are hot spots for nucleotide misincorporation (25). If pausing also induces slippage, the occurrence within the repeat tract of DNA damage that blocks DNA replication could profoundly effect TRE.Studies of replication of DNA template-primers in vitro with DNA polymerase alone are important for their potential ability to uncover mutation mechanisms. Replication of triplet-repeat tracts in vitro shows TRE (26 -29). Here, we report the effects of tract length, tract sequence, and DNA damage on TRE during DNA replication in vitro. The abasic site analog tetrahydrofuran (THF) was used as the model DNA damage lesion. A...
The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.
Elevated levels of IGF-I in the circulation are associated with increased risk for the development of prostate cancer in men, and transgenic expression of human IGF-I in mouse epithelial prostate cells results in spontaneous prostate tumorigenesis. Little, however, is known about the mechanisms involved in the IGF-I-regulated growth of prostate cells. Here, we have demonstrated that treatment with IGF-I induces the activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and the growth of human prostate cells. Stimulation with IGF-I also promoted the tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Signal relay from IGF-I to ERK requires heterotrimeric G proteins and EGFR; inhibition of Gi/o protein activation by pertussis toxin, or EGFR by tyrphostin AG1478 obliterated the ability of IGF-I to promote ERK activation. Further, treatment with pertussis toxin inhibited the IGF-I-mediated prostate cell growth. These data demonstrated the requirement of heterotrimeric G proteins in IGF-I-regulated prostate cell growth and suggest the potential utility of the G proteins as effective drug targets to combat this common cancer.
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