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.

• Authors: Ranjith P Anand, Kartik A Shah, Hengyao Niu, Patrick Sung, Sergei M Mirkin, Catherine H Freudenreich
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 40 Issue 3 Pg. 1091-105 (Feb 2012) ISSN: 1362-4962 [Electronic] England
• PMID: 21984413 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• DNA-Binding Proteins
• Proliferating Cell Nuclear Antigen
• Saccharomyces cerevisiae Proteins
• SRS2 protein, S cerevisiae
• DNA
• PIF1 protein, S cerevisiae
• Rrm3 protein, S cerevisiae
• SGS1 protein, S cerevisiae
• DNA Helicases
• RecQ Helicases

Topics

• Chromosome Breakage
• DNA (chemistry)
• DNA Helicases (metabolism, physiology)
• DNA Replication
• DNA-Binding Proteins (metabolism)
• G-Quadruplexes
• Nucleic Acid Conformation
• Proliferating Cell Nuclear Antigen (metabolism)
• RecQ Helicases (physiology)
• Repetitive Sequences, Nucleic Acid
• Saccharomyces cerevisiae Proteins (metabolism, physiology)
• Telomere (metabolism)