6 research outputs found
NUCLEOSOMES DYNAMICS AT DNA DOUBLE STRAND BREAKS
Background: DNA double-strand breaks (DSBs) are highly toxic lesions that, if not correctly repaired, can have detrimental
consequences on genome integrity and cell survival. Repair of DSBs by homologous recombination (HR) requires the
processing of DSB ends during DNA end resection, which, through the degradation of 5’-terminated strands, generates a
long stretch of single-stranded DNA (ssDNA). Therefore, resection divides the chromatin surrounding a DSB in distinct
ssDNA and dsDNA domains. The molecular composition of these domains is crucial for HR repair as well as for DNA damage
signaling and checkpoint activation. However, it was unclear whether nucleosomes, the fundamental unit of chromatin,
could be found in the ssDNA domain as well. [...
Structural mechanism of extranucleosomal DNA readout by the INO80 complex
The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80
remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to
be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated
by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker
DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be
regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits
and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes
A SAM key domain required for activation of catalytic activity of the Fun30 nucleosome remodeler
A cell cycle-regulated Slx4-Dpb11 complex promotes the resolution of DNA repair intermediates linked to stalled replication
A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81–Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11–Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81–Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81–Mms4 binding to the Dpb11–Slx4 complex. Thus, Dpb11–Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81
A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler
Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of
nucleosome remodelers involved in DNA repair and gene silencing.
These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly
understood. Using multiple sequence alignment and structure
prediction, we identify an evolutionarily conserved domain that is
modeled to contain a SAM-like fold with one long, protruding helix,
which we term SAM-key. Deletion of the SAM-key within budding yeast
Fun30 leads to a defect in DNA repair and gene silencing similar to that
of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is
able to bind nucleosomes but is deficient in DNA-stimulated ATPase
activity and nucleosome sliding and eviction. A structural model based
on AlphaFold2 prediction and verified by crosslinking-MS indicates an
interaction of the long SAM-key helix with protrusion I, a subdomain
located between the two ATPase lobes that is critical for control of
enzymatic activity. Mutation of the interaction interface phenocopies
the domain deletion with a lack of DNA-stimulated ATPase activation
and a nucleosome-remodeling defect, thereby confirming a role of the
SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of
allosteric activation for this class of enzymes