Overcoming natural replication barriers: differential helicase requirements

Abstract

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.

Genome rearrangements caused by interstitial telomeric sequences in yeast

“…Similar induction of mutagenesis was observed for (GAA) n and inverted repeats [repeat-induced mutagenesis (RIM)] (11,(13)(14)(15). All these repeats stall the replication fork progression (24)(25)(26), and the (TGTGTGGG) 15 run is a particularly potent replication block. A connection between stalled replication forks and DSBs in yeast also has been observed for CTG repeats (22) and in mec1 cells treated with hydroxyurea (27).…”

“…S5). Furthermore, fork stalling at ITSs decreases dramatically if we knock out Tof1 protein (24), which is known to preserve protein-mediated replication blocks (37).…”

Oligodeoxynucleotide Binding to (CTG) · (CAG) Microsatellite Repeats Inhibits Replication Fork Stalling, Hairpin Formation, and Genome Instability

“…In both prokaryotic and eukaryotic cells, discontinuous synthesis and ligation of the lagging strand involve molecular mechanisms more complex than the synthesis of the leading strand, suggesting that lagging-strand synthesis may be rate limiting for coupled polymerase movement on the leading and lagging arms of the replication fork (46). Indeed, polymerase stalling by hairpin-prone sequences can impede replisome progression, while enzymatic neutralization of hairpin formation has been shown to release fork stalling in S. cerevisiae (47).…”

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

“…Hybridization was performed with a probe covering the left part of the ARG2 gene, labeled by random priming [44]. Replication pausing signals and Y arc signals were quantified on a Fujifilm FLA-9000, as in [16] with slight modifications.…”

“…Slippage was supposed to occur following replication fork stalling and restart, after dissociation/reassociation of the newly-synthesized strand from its template strand [10][11][12], due to transient formation of CAG/CTG hairpin structures [13][14][15]. Replication fork pauses were shown to occur within plasmid-borne CCG/CGG, CAG/CTG and GAA/TTC triplet repeats, in bacteria, yeast and human cells [16][17][18][19][20], whereas GAA/TTC repeats were shown to transiently stall replication forks when cloned in a yeast natural chromosome [21]. No proof of replication fork stalling due to chromosomal-borne CAG/CTG repeats was ever shown.…”