Biology subjects

Yurieva, O.

Publications and source records attributed to Yurieva, O..

4 recordsLinked to original sources

bioRxiv2025

A non-catalytic role for RFC in PCNA-mediated processive DNA synthesis

The ring-shaped sliding clamp PCNA enables DNA polymerases to perform processive DNA synthesis during replication and repair. The loading of PCNA onto DNA is catalyzed by the ATPase clamp loader RFC. Using a single-molecule platform to visualize the dynamic interplay between PCNA and RFC on DNA, we unexpectedly discovered that RFC continues to associate with PCNA after loading, contrary to the conventional view. Functionally, this clamp-loader/clamp complex is required for processive DNA synthesis by polymerase {delta} (Pol{delta}), as the PCNA-Pol{delta} assembly is inherently unstable. This architectural role of RFC is dependent on the BRCT domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to DNA damage in vivo. We further showed the FEN1 flap endonuclease can also stabilize the PCNA-Pol{delta} interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions essential for DNA replication and genome maintenance.

biophysics↗

bioRxiv2021

ORC binds and remodels nucleosomes to specify MCM loading onto DNA

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c.. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with new analysis of genome-wide ORC binding, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

biophysics↗

bioRxiv2019

Single-molecule imaging of eukaryotic replisomes reveals compositional plasticity

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol DNA polymerase activity in replication and show that Pol primase and the lagging-strand Pol {delta} can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

biophysics↗

bioRxiv2017

Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

biochemistry↗