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    ABSTRACT: Mapping sites of wild-type SUMO modification is a challenging endeavour. Here we postulate that a combination of chemical derivatistation and collision-induced dissociation (CID) could be used to generate SUMO remnant diagnostic ions to aid both detection of these isopeptides and increase the analytical value of the product ion spectra required to characterize the nature and position of modification. SUMO(2/3)ylated proteins were digested with trypsin to generate isopeptides bearing TGG and QTGG isotags. The resulting digests were then dimethyl labelled followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) utilising CID in a data-dependent acquisition on a QSTAR XL. Product ion spectra were interrogated for the presence of iso-N-terminal fragment ions in addition to backbone sequence ions. The ability to diagnostically detect these isopeptides was tested by generation of co-XICs of the iso-N-terminal fragments in a semi-complex background. Dimethyl labelling facilitated the robust detection of a1', b2' & b3' (TGG isotag) and a1', b2' & b4' (QTGG isotag) ions. The abundance of both N-terminal and iso-N-terminal fragment ions, supported by dimethyl labelling, facilitated the generation of information-rich product ion spectra of these isopeptides to aid confident site assignment. Moreover, the diagnostic nature of the combined XICs of the iso-N-terminal fragments supported detection of the isopeptide signals from a semi-complex background. A combination of dimethyl labelling and CID does indeed lead to the generation of SUMO remnant isopeptide product ion spectra which are more analytically rich. This enables an improvement in characterization of both the isotag and backbone sequences and the site of modification. The diagnostic value of iso-N-terminal fragment ions allows for post-acquisition XIC interrogation to detect putative isopeptides of interest. Copyright © 2013 John Wiley & Sons, Ltd.
    No preview · Article · Sep 2013 · Rapid Communications in Mass Spectrometry
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    ABSTRACT: DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.
    Full-text · Article · Mar 2013 · Cell Reports
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    ABSTRACT: Background: The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation. Results: Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol ε (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol ε to interact with the replisome after initiation. Conclusions: These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.
    Preview · Article · Mar 2013 · Current biology: CB
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