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1.
EMBO J ; 42(22): e114334, 2023 Nov 15.
Article in English | MEDLINE | ID: mdl-37781931

ABSTRACT

Sequences that form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play various physiological roles. However, they can also interfere with replication and threaten genome stability. Multiple lines of evidence suggest G4s inhibit replication, but the underlying mechanism remains unclear. Moreover, evidence of how iMs affect the replisome is lacking. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM arrest DNA replication. Direct single-molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. Combined genetic and biophysical characterisation establishes that structure stability and probability of structure formation are key determinants of replisome arrest. Mechanistically, replication arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Rrm3, Sgs1, Chl1 or Hrq1. Altogether, we provide a mechanism for quadruplex structure formation and resolution during replication and highlight G4s and iMs as endogenous sources of replication stress.


Subject(s)
DNA , G-Quadruplexes , Humans , Genome, Human , Nucleotidyltransferases , DNA Replication
2.
Elife ; 112022 12 19.
Article in English | MEDLINE | ID: mdl-36533901

ABSTRACT

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.


Subject(s)
Bacteriophages , Escherichia coli Proteins , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Bacteriophages/genetics , Bacteriophages/metabolism , Exodeoxyribonuclease V/genetics , DNA/metabolism , DNA, Single-Stranded/metabolism , Recombinases/metabolism , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , DNA, Bacterial/metabolism
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