Journal of Molecular Biology
Volume 425, Issue 2, 23 January 2013, Pages 232-243
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Mechanism of Replicative DNA Polymerase Delta Pausing and a Potential Role for DNA Polymerase Kappa in Common Fragile Site Replication

https://doi.org/10.1016/j.jmb.2012.11.016Get rights and content

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 (< 40 base 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.

Highlights

► Mechanisms by which CFS repeat sequences affect Pols are unknown. ► Polymerase δ pauses at microsatellite and palindromic sequences within CFSs. ► Polymerase δ pausing is due to sequence-specific polymerase dissociation. ► Polymerase κ displays more efficient synthesis of CFS repeat sequences. ► Polymerase κ may be required for replication of CFS repeat sequences in cells.

Introduction

Genomic instability is a driving force of tumor evolution and numerous biochemical mechanisms are required for accurate genome maintenance and duplication in normal cells. Chromosomal fragile sites are genomic loci that are prone to instability in the form of gaps and breaks under specific cellular conditions.1 Common fragile sites (CFSs) exist in all individuals as normal chromosomal components but undergo breakage at increased frequencies upon treatment of cells with specific chemicals, such as aphidicolin.[1], [2] Of clinical significance, breakpoints of chromosomal translocations and deletions in several tumor types have been mapped to CFSs.[1], [3], [4] A large number of studies have shown that loss of specific replication, cell cycle checkpoint, and DNA repair proteins leads to CFS instability.[1], [5], [6], [7] These findings suggest that maintenance of CFS stability in normal cells requires DNA replication checkpoint and repair responses. Several non-mutually exclusive models have been proposed to explain the propensity for specific chromosomal breakage within CFS regions. These models include, but are not limited to, late timing of CFS replication,8 paucity of replication initiation events within CFSs,9 failure to activate additional replication origins under stress,10 altered epigenetic patterns,11 and collision of the transcription and replication machinery within long genes.12

Ectopic integration of ~ 140- to 150-kb FRA3B sequences led to increased aphidicolin-induced gaps and breaks at the new chromosomal location.13 In addition to studies showing altered DNA replication initiation within CFSs, non-B DNA secondary structures, such as hairpins, cruciforms, and large AT-rich flexibility regions, have been hypothesized to hinder elongation by the replication machinery, thus contributing to slowed replication at CFSs.[1], [7], [14], [15] Clusters of AT-rich flexibility peaks may act as sinks for the superhelical density generated ahead of the replication fork, thereby hindering efficient topoisomerase activity and inhibiting replication fork progression.14 Experimentally, an [AT/TA]34 microsatellite derived from FRA16D and predicted to form a large hairpin structure was shown to induce replication fork stalling and chromosomal fragility in a Saccharomyces cerevisiae model.16 In human cells, replication blockage was also shown to occur preferentially near AT-rich regions within FRA16C, even in the absence of aphidicolin, and the rate of replication fork progression was found to be decreased within FRA16C, compared to the whole genome.10 While these experiments demonstrate that AT-rich regions within CFSs impede the replication fork, the degree to which DNA synthesis by the replicative polymerases is affected has not been extensively investigated. We previously showed that a mononucleotide [A]28 repeat derived from a region within FRA16D contributed to the pausing of DNA synthesis in vitro by the replicative polymerases α and δ [DNA polymerase (Pol) δ].17 In a recent genome-wide statistical modeling study of genomic features distinguishing CFS from non-fragile regions, we found that mononucleotide A/T microsatellite coverage (associated with Alu repeats), low-complexity AT coverage, and DNA flexibility are significant predictors of CFS regions in alternative models.18

Specialized Pols may be required for synthesis of naturally occurring alternative DNA structures that replicate late in S phase, such as those found within CFSs. Depletion of Pol η leads to increased chromosomal breakage at FRA7H, a slight delay in S phase, and a decrease in the number of late S phase replication foci.19 Depletion of either Pol η or Pol κ results in increased double-stranded breaks upon ectopic integration of constructs containing non-B DNA elements.20 We recently showed that Pol κ displays significantly less synthesis termination within mononucleotide [T]1121 and dinucleotide [GT]10 microsatellites, compared to replicative polymerases.22 These findings led us to hypothesize that Pol κ may also be important for maintaining efficient replication through CFS sequences.

The goals of this study were to understand (1) the mechanisms limiting replicative Pol δ DNA synthesis through specific sequence elements contained within CFS regions and (2) the potential contribution of the specialized Pol κ to efficient CFS DNA synthesis. We demonstrate that Pol δ undergoes significant pausing at three types of repetitive sequence elements, AT-rich microsatellites (A/Tn and AT/TAn) and quasi-palindromes (QPs), in a length-dependent manner. Mechanistically, we show that Pol δ pause sites within CFS sequences represent sites of enzyme dissociation from the DNA template. Finally, we show that DNA synthesis by Pol κ is significantly more efficient through these types of repetitive elements found within CFS templates. Based on our in vitro findings, we propose that the specialized Pol κ might function in replication of repetitive DNA sequences in order to maintain CFS stability.

Section snippets

Polymerase pausing assay

We analyzed the efficiency of polymerase DNA synthesis using templates corresponding to CFS sequences and an in vitro primer extension assay that models DNA synthesis on the lagging strand of the replication fork. Single-stranded DNA (ssDNA) templates that contain ~ 120–170 base pairs derived from the reference sequences for FRA16D or FRA3B were constructed (Table 1 and Fig. 1). These templates harbor repetitive sequence elements identified using the Non-B DNA Database

Discussion

Genome instability is a hallmark of tumorigenesis, and chromosomal rearrangements in tumor cells frequently occur at CFSs. Determining the genomic features that contribute to CFS instability and the replication proteins that maintain stability in normal cells will expand our understanding of how genome instability arises during tumorigenesis. Here, we investigated the DNA sequence elements and Pols that affect the efficiency of DNA synthesis, in order to gain insight into whether the elongation

Replication proteins

Recombinant human four-subunit Pol δ and PCNA were purified using baculovirus expression systems, as described previously.52 Human Pol κ and additional PCNA were purchased from Enzymax (Lexington, KY). Yeast RFC was a generous gift from Dr. Gregory D. Bowman.

Vector constructs

Oligonucleotides corresponding to FRA16D or FRA3B regions (derived from GenBank accessions AF217490 and 183583557, respectively) were purchased from IDT (Coralville, IA). Double-stranded DNA for cloning was generated for each

Acknowledgements

This work was supported by the National Institutes of Health (grant number CA100060 to K.A.E. and grant number GM313973 to M.Y.L) and the Gittlen Cancer Research Foundation (Penn State Hershey Medical Center). This project was also funded, in part, under a grant from the Pennsylvania Department of Health using Tobacco CURE Funds (SAP# 4100042746 to K.A.E.). The Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. The authors thank Suzanne

Glossary

Common fragile sites:
Genomic loci that undergo a high frequency of chromosomal breakage under specific cellular conditions of replication stress.
Microsatellites:
DNA sequences of 1–6 base pairs repeated in tandem.
Quasi-palindromes:
Repetitive DNA sequence elements composed of an imperfect palindrome (inverted repeat sequence) separated symmetrically by an intervening sequence. DNA hairpins predicted to form within quasi-palindromes: stem structures are formed by intramolecular base pairing within

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