NYMC Faculty Publications

Mechanism of Replicative DNA Polymerase Delta Pausing and a Potential Role for DNA Polymerase Kappa in Common Fragile Site Replication

Author Type(s)

Faculty

DOI

10.1016/j.jmb.2012.11.016

Journal Title

Journal of Molecular Biology

First Page

232

Last Page

243

Document Type

Article

Publication Date

1-23-2013

Department

Biochemistry and Molecular Biology

Keywords

Chromosome Fragile Sites, DNA, DNA Polymerase III, DNA Replication, DNA-Directed DNA Polymerase, Humans, Molecular Sequence Data, Proliferating Cell Nuclear Antigen

Disciplines

Medicine and Health Sciences

Abstract

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 ~75-100% self-homology, separated by 3-9 bases of intervening sequences. Analysis of DNA synthesis progression by human Pol δ demonstrated significant synthesis perturbation both at [A](n) and [TA](n) repeats in a length-dependent manner and at short (pairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ 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 δ 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 κ.

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