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摘要
Common fragile sites (CFSs) are hot spots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFSs to the inhibition of DNA synthesis by replicative and specialized DNA polymerases (Pols). The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of similar to 75-100% self-homology, separated by 3-9 bases of intervening sequences. Analysis of DNA synthesis progression by human Pol delta demonstrated significant synthesis perturbation both at [A](n) and [TA](n) repeats in a length-dependent manner and at short (%26lt;40 base pairs) QP sequences. DNA synthesis by the Y-family polymerase kappa was significantly more efficient than Pol delta through both types of repetitive elements. Using DNA trap experiments, we show that Pol delta pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative Pol pausing and dissociation. Our finding that Pol delta dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase kappa.
- 出版日期2013-1-23
