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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
3.
EMBO J ; 40(5): e107037, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33555053

ABSTRACT

The high-fidelity replicative DNA polymerases, Pol ε and Pol δ, are generally thought to be poorly equipped to replicate damaged DNA. Direct and complete replication of a damaged template therefore typically requires the activity of low-fidelity translesion synthesis (TLS) polymerases. Here we show that a yeast replisome, reconstituted with purified proteins, is inherently tolerant of the common oxidative lesion thymine glycol (Tg). Surprisingly, leading-strand Tg was bypassed efficiently in the presence and absence of the TLS machinery. Our data reveal that following helicase-polymerase uncoupling a switch from Pol ε, the canonical leading-strand replicase, to the lagging-strand replicase Pol δ, facilitates rapid, efficient and error-free lesion bypass at physiological nucleotide levels. This replicase switch mechanism also promotes bypass of the unrelated oxidative lesion, 8-oxoguanine. We propose that replicase switching may promote continued leading-strand synthesis whenever the replisome encounters leading-strand damage that is bypassed more efficiently by Pol δ than by Pol ε.


Subject(s)
DNA Damage , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Thymine/analogs & derivatives , DNA-Directed DNA Polymerase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Thymine/chemistry
4.
EMBO J ; 35(9): 961-73, 2016 05 02.
Article in English | MEDLINE | ID: mdl-26912723

ABSTRACT

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.


Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Minichromosome Maintenance Complex Component 4/metabolism , Minichromosome Maintenance Complex Component 6/metabolism , Phosphopeptides/metabolism , Protein Serine-Threonine Kinases/metabolism , Replication Origin , Saccharomyces cerevisiae Proteins/metabolism , DNA Replication , Nuclear Proteins/metabolism , Protein Binding , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development
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