Replication stalling and DNA microsatellite instability

Abstract

Microsatellites are short, tandemly repeated DNA motifs of 1-6 nucleotides, also termed simple sequence repeats (SRSs) or short tandem repeats (STRs). Collectively, these repeats comprise approximately 3% of the human genome Subramanian et al. (2003), Lander and Lander (2001) [1,2], and represent a large reservoir of loci highly prone to mutations Sun et al. (2012), Ellegren (2004) [3,4] that contribute to human evolution and disease. Microsatellites are known to stall and reverse replication forks in model systems Pelletier et al. (2003), Samadashwily et al. (1997), Kerrest et al. (2009) [5-7], and are hotspots of chromosomal double strand breaks (DSBs). We briefly review the relationship of these repeated sequences to replication stalling and genome instability, and present recent data on the impact of replication stress on DNA fragility at microsatellites in vivo.

Fig. 1

Hypothetical models of replication-dependent instability. Red arrows indicate nascent DNA repeat sequences. A. 3′ end slippage. Dissociation of the DNA polymerase, partial denaturation of the nascent DNA 3′ end, hairpin formation, and out-of-register reannealing to the template may lead to expansion after another round of replication. B. 5′ flap hairpin. Displacement synthesis can allow hairpin formation in the nascent DNA, reannealing, and ligation to the upstream Okazaki fragment. C. Hairpin isomerization. A template strand hairpin causes 3′ end slippage and may allow equilibration of the hairpin. Reannealing of the nascent 3′ end and Okazaki fragment ligation stabilize the contraction in the nascent DNA. D. Fork reversal. Polymerase stalling in repetitive DNA may allow fork reversal and hairpin formation in leading strand nascent DNA (upper panel) or lagging strand nascent DNA after Okazaki fragment dissociation and repriming (lower panel). E. Template switching. Polymerase stalling at a non-B structure causes the leading strand polymerase to use nearby lagging strand nascent DNA as a template. Reannealing of the nascent strand to the leading strand template can lead to hairpins in the template or nascent DNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Oligodeoxynucleotide inhibition of hairpin formation. A. Schematicof the CTG-directed zinc finger nuclease (ZFN_CTG) homodimer binding to a CTG hairpin. Note that dimerization of the FOK1 nuclease domain (large oval) is required for cleavage, therefore a ZFN_CTG homodimer can cleave a CTG hairpin but not CTG/CAG dsDNA [64]. B. Inhibition of hairpin formation by (CAG)₇ in cells containing the ectopic c-myc core origin and (CTG)₄₅ in the lagging strand template. Lanes 1 to 4, mock ODN treatment of cells expressing ZFP_CTG; lanes 5 to 8, mock ODN treatment of cells expressing ZFN_CTG; lanes 9 to 12, (CTG)₇ treatment of cells expressing ZFP_CTG; lanes 13 to 16, (CAG)₇ treatment of cells expressing ZFN_CTG. The 270 bpPCR product [(CTG/ CAG)₄₅ progenitor allele] is indicated with an arrow. The lower-mobility shadow bands observed above the 270 bp amplification product of the (CTG/CAG)₄₅ progenitor sequence are slipped-strand structures formed in vitro during PCR reannealing. Bands migrating faster than the progenitor product are the results of in vivo ZFN_CTG cleavage. C. Model for hairpin inhibition by the (CAG)₇ ODNs. D. Inhibition of hairpin formation by (CTG)₇ in (CAG)₄₅ cells. Lanes 1 to 4, mock ODN treatment of cells expressing ZFP_CAG; lanes 5 to 8, mock ODN treatment of cells expressing ZFN_CAG; lanes 9 to 12, (CTG)₇ treatment of cells expressing ZFP_CAG; lanes 13 to 16, (CTG)₇ treatment of cells expressing ZFN_CAG. E. Model for hairpin inhibition by (CTG)₇ in (CAG)₄₅ cells. In panels C and E, ODNs are depicted as multiple short bars, repeated CTG or CAG sequences are depicted in red, and ZFNs are depicted in blue. Reprinted with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

FANCJ knockdown leads to loss of ectopic CTG/CAG microsatellite signals in (CTG/CAG)₁₀₂ cells under replication stress. A. Western blots. Whole cell extracts were isolated after treatment of cells with siControl or siFANCJ for a total of five transfections, and 0.2 µM aphidicolin, or parallel untreated cultures and immunoblotted for FANCJ. B. Duplex small pool PCR with primers spanning a non-repeat internal PCR control site and the ectopic (CAG)₁₀₂ repeats (left) or (CTG)₁₀₂ repeats (right). Notice the loss of the ectopic site repeat PCR band in cells knocked down for FANCJ and treated with APH. Reprinted with permission.

Fig. 4

FANCJ null patient cells treated with aphidicolin are prone to microsatellite signal loss at multiple endogenous sites. A–L. Duplex spPCR across endogenous repeated sequences in DNA from FANCJ null patient fibroblasts with or without aphidicolin (0.2 µM) treatment. Reprinted with permission.

Fig. 5

Chromosome fragility at an ectopic CTG microsatellite. A. DF myc·CTG cells are a clonal HeLa cell line containing a single copy integrant of the c-myc core replication origin [183] next to a CTG/CAG microsatellite, flanked by red (Tomato)and green (eGFP) marker genes. B,D,F.Flow cytometry of untreated DF2 myc, DF2 myc.CTG₂₃, and DF2 myc.CTG₁₀₀ cells. C, E, G. Flow cytometry of untreated DF2 myc, DF2 myc.CTG₂₃, and DF2 myc.CTG₁₀₀ cells treated with 0.2 mM HU for six days and allowed to recover in drug-free medium for four days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)