Looping out of control: R-loops in transcription-replication conflict.

Transcription-replication conflict is a major cause of replication stress that arises when replication forks collide with the transcription machinery. Replication fork stalling at sites of transcription compromises chromosome replication fidelity and can induce DNA damage with potentially deleterious consequences for genome stability and organismal health. The block to DNA replication by the transcription machinery is complex and can involve stalled or elongating RNA polymerases, promoter-bound transcription factor complexes, or DNA topology constraints.

A transcription-based approach to purify R-loop-containing plasmid DNA templates in vitro.

To study the direct effects of R-loops on DNA replication and other DNA-templated processes in vitro, R-loop-containing DNA templates need to be prepared efficiently and to near homogeneity. Here, we describe a simple transcription-based approach to form R-loops on plasmid DNA templates in vitro. We detail steps to transcribe a DNA sequence element with a high propensity to form co-transcriptional R-loops using T7 RNA polymerase.

Multistep loading of a DNA sliding clamp onto DNA by replication factor C.

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 (ss)/double-stranded DNA (dsDNA) 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 ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy.

CMG helicase activity on G4-containing DNA templates.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in G-rich regions of the genome and threaten genome stability by interfering with DNA replication. However, the underlying mechanisms are poorly understood. We have recently found that G4s can stall eukaryotic replication forks by blocking the progression of replicative DNA helicase, CMG.

Yeast ORC sumoylation status fine-tunes origin licensing.

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear.

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 of embryo size, pre-gastrulation 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.

Mobile origin-licensing factors confer resistance to conflicts with RNA polymerase.

Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during the G1 phase and where synthesis begins in S phase. However, the temporal delay between phases exposes the replisome assembly pathway to potential disruption prior to replication. Using multicolor, single-molecule imaging, we systematically study the consequences of encounters between actively transcribing RNA polymerases (RNAPs) and replication initiation intermediates in the context of chromatin.

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

Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state.

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

Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In , 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.

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.

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 extension (NTE) 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.

DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis.

DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast.

Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks.

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA.

Cdt1 stabilizes an open MCM ring for helicase loading.

ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands.

Eukaryotic replication origins: Strength in flexibility.

The eukaryotic replicative DNA helicase, Mcm2-7, is loaded in inactive form as a double hexameric complex around double-stranded DNA. To ensure that replication origins fire no more than once per S phase, activation of the Mcm2-7 helicase is temporally separated from Mcm2-7 loading in the cell cycle. This 2-step mechanism requires that inactive Mcm2-7 complexes be maintained for variable periods of time in a topologically bound state on chromatin, which may create a steric obstacle to other DNA transactions.

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading.

Origin plasticity during budding yeast DNA replication in vitro.

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre-replicative complexes (pre-RCs) license origins by loading Mcm2-7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2-7 DNA helicase.

ATPase-dependent quality control of DNA replication origin licensing.

The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6.

Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP.

Eukaryotic DNA replication control: lock and load, then fire.

The initiation of chromosomal DNA replication involves initiator proteins that recruit and load hexameric DNA helicases at replication origins. This helicase loading step is tightly regulated in bacteria and eukaryotes. In contrast to the situation in bacteria, the eukaryotic helicase is loaded in an inactive form.

CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex.

Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation.

DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding.

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction.