Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.     

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

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Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.     

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2–7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2–7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2–7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.     

Divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on modulating the loading of the MCM helicase onto the origins of the hyperthermophilic archaeon Sulfolobus solfataricus P2

The Cdc6 protein has been suggested as a loader for the eukaryotic MCM helicase. Archaeal replication machinery represents a core version of that in eukaryotes. In the current work, three eukaryotic Orc1/Cdc6 homologs (SsoCdc6-1, -2, and -3) from crenarchaeon Sulfolobus solfataricus were shown to have totally different effects on the interactions with SsoMCM helicase. SsoCdc6-2 stimulates the binding of the SsoMCM onto the origin DNA, but SsoCdc6-1 and SsoCdc6-3 significantly inhibit the loading activities, and these inhibitive effects can not be reversed by the stimulation of SsoCdc6-2. Using pull-down assays, we showed that three SsoCdc6 proteins interacted physically with the SsoMCM. Furthermore, the C-terminal domains of SsoCdc6 proteins were shown to physically and functionally affect the interactions with SsoMCM. This is the first report on the divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on regulating the loading of the MCM helicase onto the origins in the archaeon.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

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Regulation and roles of Cdc7 kinase under replication stress

Cdc7 (cell division cycle 7) kinase together with its activation subunit ASK (also known as Dbf4) play pivotal roles in DNA replication and contribute also to other aspects of DNA metabolism such as DNA repair and recombination. While the biological significance of Cdc7 is widely appreciated, the molecular mechanisms through which Cdc7 kinase regulates these various DNA transactions remain largely obscure, including the role of Cdc7-ASK/Dbf4 under replication stress, a condition associated with diverse (patho)physiological scenarios. In this review, we first highlight the recent findings on a novel pathway that regulates the stability of the human Cdc7-ASK/Dbf4 complex under replication stress, its interplay with ATR-Chk1 signaling, and significance in the RAD18-dependent DNA damage bypass pathway. We also consider Cdc7 function in a broader context, considering both physiological conditions and pathologies associated with enhanced replication stress, particularly oncogenic transformation and tumorigenesis. Furthermore, we integrate the emerging evidence and propose a concept of Cdc7-ASK/Dbf4 contributing to genome integrity maintenance, through interplay with RAD18 that can serve as a molecular switch to dictate DNA repair pathway choice. Finally, we discuss the possibility of targeting Cdc7, particularly in the context of the Cdc7/RAD18-dependent translesion synthesis, as a potential innovative strategy for treatment of cancer.     

Genome Protection by the 9-1-1 Complex Subunit HUS1 Requires Clamp Formation,DNA Contacts,and ATR Signaling-independent Effector Functions

The RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and repair at DNA damage sites. In this study, we elucidated HUS1 functional residues that drive clamp assembly, DNA interactions, and downstream effector functions. First, we mapped a HUS1-RAD9A interface residue that was critical for 9-1-1 assembly and DNA loading. Next, we identified multiple positively charged residues in the inner ring of HUS1 that were crucial for genotoxin-induced 9-1-1 chromatin localization and ATR signaling. Finally, we found two hydrophobic pockets on the HUS1 outer surface that were important for cell survival after DNA damage. Interestingly, these pockets were not required for 9-1-1 chromatin localization or ATR-mediated CHK1 activation but were necessary for interactions between HUS1 and its binding partner MYH, suggesting that they serve as interaction domains for the recruitment and coordination of downstream effectors at damage sites. Together, these results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance.     

Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

Functional differentiation and cooperative interaction between two eukaryote-like archaeal Orc1/Cdc6 proteins on the replication origin

The DNA replication apparatus of archaea represents a core version of that in eukaryotes. Archaeal Orc1/Cdc6s can be an integral component in the replication machineries cooperatively regulating DNA replication. We investigated the DNA-binding activities of two eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1 and -2) and interactions between them on the different structural duplex DNA substrates derived from oriC1 of Sulfolobus solfataricus. The results showed that two Orc1/Cdc6 proteins stimulated mutual DNA-binding activities at lower concentrations and formed bigger SsoCdc6-1/SsoCdc6-2/DNA complex at higher concentrations. Furthermore, SsoCdc6-2 stimulated the DNA-binding activity of SsoMCM and demonstrated a high affinity to the 5-forked DNA. In contrast, SsoCdc6-1 inhibited the binding of SsoMCM and demonstrated better affinity to the sequence-specific blunt DNA substrate. Finally, we found that the two proteins physically interacted with each other and with SsoMCM. Thus, the two Orc1/Cdc6 proteins were functionally different, but they may keep the coordinated interaction on the replication origin.     

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.     

Ubiquitin-specific Peptidase 20 Regulates Rad17 Stability,Checkpoint Kinase 1 Phosphorylation and DNA Repair by Homologous Recombination

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.     

Chromatin Remodeling and Immediate Early Gene Activation by SLFN11 in Response to Replication Stress

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