Biology subjects

Remus, D.

Publications and source records attributed to Remus, D..

9 recordsLinked to original sources

bioRxiv2024

Cryo-EM structures of G4-stalled CMG reveal inchworm mechanism of DNA translocation

DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we have characterized fork collisions with preformed G4s in the parental DNA using fully reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by blocking the CMG helicase. An ensemble of high-resolution cryo-EM structures of stalled yeast and human CMG complexes reveals that the G4 is fully folded and lodged inside the CMG central channel. The stalled CMG is conformationally constrained and arrests in the transition between translocation states. Unexpectedly, our analysis suggests that CMG employs an unprecedented inchworm mechanism to translocate on DNA. These findings illuminate the eukaryotic replication fork mechanism under both normal and perturbed conditions.

biochemistry↗

bioRxiv2022

A hypomorphic mutation in Pold1 disrupts the coordination of embryo size expansion and morphogenesis during gastrulation

Formation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations, pre-gastrulation stage mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle related genes frequently lead to cell cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase {delta}. Impaired cell proliferation in the tyrn mutant leaves anterior-posterior patterning unperturbed during gastrulation but results in an overall reduction in embryo size and in severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth. Summary statementPold1 hypomorphic mutation caused reduced size and abnormal morphology of gastrulating mouse embryos, supporting the importance of coordinated embryo size, lineage specification and tissue morphogenesis for normal embryogenesis.

developmental biology↗

bioRxiv2022

Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an elevator-with-an-operator mechanism.

molecular biology↗

bioRxiv2022

Multistep loading of PCNA onto DNA by RFC

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3 end (3 ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATP{gamma}S and 3 ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

molecular biology↗

bioRxiv2021

Mechanisms of loading and release of the 9-1-1 checkpoint clamp

5 single-stranded/double-stranded DNA serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1s recruitment to 5 junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-RFC, in complex with 9-1-1 and a 5 junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1s recruitment to 5 junctions and reveal new principles of sliding clamp loading.

biophysics↗

bioRxiv2021

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14 and Sic1 for origin licensing in S. cerevisiae

Cdc6, a subunit of the pre-replicative complex, contains multiple regulatory Cdk1 consensus sites, SP or TP motifs. In S. cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S-phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the Cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, the Cdk1 inhibitor, Sic1, releases Clb2{middle dot}Cdk1{middle dot}Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing is promoted by the attenuation of distinct CDK-dependent Cdc6 inhibitory mechanisms.

cell biology↗

bioRxiv2021

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

biochemistry↗

bioRxiv2020

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

biochemistry↗

bioRxiv2019

Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol {varepsilon} variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol {delta} from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol {delta} by Pol {varepsilon} restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

molecular biology↗