Helicase Complex Research Articles

The Fate of Two Unstoppable Trains After Arriving Destination: Replisome Disassembly During DNA Replication Termination.

In eukaryotes, the perfect duplication of the chromosomes is executed by a dynamic molecular machine called the replisome. As a key step to finishing DNA replication, replisome disassembly is triggered by ubiquitylation of the MCM7 subunit of the helicase complex CMG. Afterwards, the CDC48/p97 “unfoldase” is recruited to the ubiquitylated helicase to unfold MCM7 and disassemble the replisome. Here we summarise recently discovered mechanisms of replisome disassembly that are likely to be broadly conserved in eukaryotes. We also discuss two crucial questions that remain to be explored further in the future. Firstly, how is CMG ubiquitylation repressed by the replication fork throughout elongation? Secondly, what is the biological significance of replisome disassembly and what are the consequences of failing to ubiquitylate and disassemble the CMG helicase?

Abstract 1277: Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells

Abstract Introduction: CDC7 is a serine/threonine protein kinase that phosphorylates the MCM2-7 helicase complex, a required step in DNA replication initiation. CDC7 has emerged as an attractive target for cancer treatment because of high expression in a number of tumors (e.g. ovarian, lung, and oral) which is thought to be linked to their proliferative capacity and ability to bypass DNA damage responses. Consistent with this, disruption of CDC7 activity in cancer cells results in delayed DNA replication, mitotic abnormalities and cell death whereas non-transformed, p53 wildtype cells are protected from cytotoxicity due to G1 cell cycle arrest. Due to the low ATP Km of CDC7, very potent inhibitor molecules are required to effectively block CDC7 activity and drive cancer cells into apoptosis. We have identified novel potent and selective CDC7 inhibitors targeting the ATP binding site that are active in biophysical, biochemical and cellular assays as well as in vivo CDX models. Results: Our lead compounds show potent picomolar (pM) inhibition of CDC7 in a biochemical kinase activity assay, pM affinity in SPR assay and complete inhibition of MCM2 (S53) phosphorylation in COLO205, A427, MV-4-11 and SW48 cancer cell lines. In a broad kinase selectivity panel, the novel inhibitors showed good selectivity for CDC7 kinase. Mechanistic studies show that our CDC7 inhibitors induced apoptosis, disrupted DNA replication and cell cycle dynamics with accumulation of polyploid cells after 48 h of treatment of cancer cells with minimal effects on human fibroblast cell lines. Our compounds have shown potent anti-proliferative and cytotoxic effects in a panel of more than a 100 cancer cell lines of varying origin including COLO205, SW48, A427, MOLM-13, and SUM149. Comparison of CDC7 inhibitors with other oncology drugs in a panel of cancer cell lines revealed a unique mechanism of action. In vivo, our compounds reduced tumor cell MCM2 (S53) phosphorylation in the mouse COLO205 xenograft model and showed strong tumor growth inhibition. We have also examined the effect of CDC7 inhibitors on cancer cell proliferation in combination with other anti-cancer agents, including other DNA damage response (DDR) targeting agents. Conclusions: We have identified novel potent ATP-competitive CDC7 inhibitors that show target engagement in cells and CDX tumors and have shown strong inhibition of cancer cell proliferation in vitro and tumor growth in vivo. CDC7 inhibitors show promise for use in combination with other targeted therapies for the treatment of cancers of varying origin. Citation Format: Lyuben Tsvetkov, Adam Levinson, Xianhai Huang, Sayan Mondal, Jeff Bell, Lin Tang, Robert Pelletier, Karen Dingley, Nick Boyles, Jackson Chief Elk, Leah Frye, Alan Futran, Phani Ghanakota, Jeremy Greenwood, George Lai, Sarah Silvergleid, Wu Yin, Hamish Wright, Karen Akinsanya, Wayne Tang, Kristian Jensen. Discovery of novel CDC7 inhibitors that disrupt cell cycle dynamics and show anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1277.

Binding of eukaryotic initiation factor 3 (eIF3) to Barley Yellow Dwarf Virus (BYDV) untranslated regions (UTRs)

Viruses remain a major threat to human health and the global food sources. Viruses are obligate parasites that use their host's cellular machinery to translate their proteins, usually employing unique non-canonical translation mechanisms to outcompete the host cell mRNA for this machinery. BYDV is a positive-strand RNA virus that lacks the canonical 5ʹ 7-methylguanosine cap and a poly-A tail in its mRNA, instead utilizing a CITE (cap-independent translation element) in the 3ʹ UTR, termed as the 3ʹ BTE (BYDV like CITE). The BYDV 3ʹ BTE is 105-nt long and has a cruciform secondary structure with three stem loops SL-I, SL-II, SL-III and a base stem (SL-IV). BTE has been shown to use the host cell's eukaryotic translation initiation factor (eIF) 4G, eIF4A, eIF4B and ATP (an active helicase complex) to recruit the 40S ribosomal subunit. This 3ʹ BTE-recruited translation initiation complex must transfer to the 5ʹ end of the mRNA for scanning, AUG detection, and translation elongation to proceed. The 5ʹ UTR of BYDV is also structured, consisting of four stem loops, (SL-A, B, C and D), and the 3ʹ to 5ʹ transfer process is aided by a relatively weak, long-distance, ‘kissing-loop’, base pairing interaction between stem-loops in the 5ʹ and 3ʹ ends of BYDV. Recently, we showed that eIF3 plays a crucial role in this transfer by simultaneously binding to both the BYDV 5ʹ and 3ʹ UTRs. These binding interactions were found to be helicase dependent, and the UTRs did not compete for eIF3 binding, suggesting the presence of a transient complex. eIF3 has been shown to interact with both the 5ʹ IRES (Internal ribosome entry site) and 3ʹ UTR of Hepatitis C Virus (HCV) simultaneously. In HCV-like IRESs, the eIF3 and the IRES binding site on the 40S subunit has been shown to overlap, requiring structural rearrangements and dislocation of eIF3 for IRES to bind. In BYDV, we expect similar rearrangements in the UTRs for eIF3 to successfully transfer the 40S complex from the 3ʹ to the 5ʹ end. To understand this macromolecular complex consisting of eIF3, the helicase complex, and the BYDV UTRs, we used a fusion RNA oligo comprising the BYDV 5ʹ UTR and 3ʹ BTE linked by a 12-base RNA linker and performed fluorescence anisotropy-based binding studies to eIF3. Interestingly, the helicase complex was not required for eIF3 binding to the Fusion oligo. SHAPE footprinting analyses performed on the fusion oligo showed that the presence of all helicase factors shifts the eIF3 binding site on the RNA closer to the kissing loop interaction between the UTRs. These data suggest that the helicase complex is not required for binding if the UTRs are in close proximity, but it does aid in rearranging the UTR RNAs to accommodate optimum eIF3 binding for eventual transfer of the 40S complex from the 3ʹ to the 5ʹ end of BYDV.

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design.

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

Site-specific and substrate-specific control of accurate mRNA editing by a helicase complex in trypanosomes.

Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited “junction” regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5′ positions but increased total editing at examined A6 3′ positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5′ nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3′-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3′–5′ progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3′ half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss.

Distinct and sequential re-replication barriers ensure precise genome duplication.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.

MCM8IP activates the MCM8-9 helicase to promote DNA synthesis and homologous recombination upon DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.

The Licensing Factor Cdt1 Links Cell Cycle Progression to the DNA Damage Response.

The maintenance of genome integrity is essential for cellular survival and propagation. It relies upon the accurate and timely replication of the genetic material, as well as the rapid sensing and repairing of damage to DNA. Uncontrolled DNA replication and unresolved DNA lesions contribute to genomic instability and can lead to cancer. Chromatin licensing and DNA replication factor 1 (Cdt1) is essential for loading the minichromosome maintenance 2-7 helicase complex onto chromatin exclusively during the G1 phase of the cell cycle, thus limiting DNA replication to once per cell cycle. Upon DNA damage, Cdt1 rapidly accumulates to sites of damage and is subsequently poly-ubiquitinated by the cullin 4-RING E3 ubiquitin ligase complex, in conjunction with the substrate recognition factor Cdt2 (CRL4Cdt2), and targeted for degradation. We here discuss the cellular functions of Cdt1 and how it may interlink cell cycle regulation and DNA damage response pathways, contributing to genome stability.

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links.

DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

Allosteric Effects of RecB Nuclease Domain on RecBCD-DNA Interactions

E. coli RecBCD is crucial in initiating repair of double stranded (ds) DNA breaks. It is a heterotrimeric helicase and nuclease complex possessing two ATPase motors, RecB and RecD, and a regulatory subunit without ATPase activity, RecC. The RecB subunit contains a 30kDa nuclease domain (RecBNuc) that, according to published structural data, is situated over 60Å away from the site of dsDNA binding. Surprisingly, we have shown that deletion of RecBNuc to form RecBΔNucCD affects its dsDNA unwinding properties. The mechanism by which RecBNuc influences RecBCD dsDNA unwinding is unclear. To probe this further, we examined how deletion of RecBNuc affects the thermodynamics of RecBCD binding to dsDNA ends using isothermal titration calorimetry (ITC) and fluorescence titrations. RecBCD can melt 6 base pairs upon binding a blunt ended DNA in an ATP-independent, but Mg2+-dependent reaction. We observe an unfavorable ΔH for binding of both RecBCD and RecBΔNucCD to blunt ended DNA, likely reflecting the cost of melting the 6 base pairs. RecBΔNucCD binds to dsDNA ends with much higher affinity than RecBCD, which is driven primarily by a more favorable ΔS°. Previous studies showed that RecBCD has optimal binding affinity for a dsDNA end possessing 3’dT6 and 5’dT10 ssDNA tails. Here, we present thermodynamic data (ΔG°, ΔH and TΔS°) showing a continued increase in favorable ΔH for binding dsDNA ends possessing ssDNA tails longer than 3’dT6 and 5’dT10. However, these more favorable ΔH values are compensated by unfavorable TΔS° values. This suggests that RecBCD makes contacts with dsDNA ends possessing ssDNA tails longer than 3’dT6 and 5’dT10, which was unexpected based on previous data. This ssDNA tail length effect differs for RecBΔNucCD. These data suggest that RecBNuc regulates RecBCD dsDNA interactions through long-range allosteric effects. (Supported by NIH grant GM045948.)

Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism.

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction.

NK cell defects in X-linked pigmentary reticulate disorder.

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3–CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.

Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase

Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme-DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

Abstract LB-288: CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics

Abstract DNA replication is a tightly regulated process required for faithful duplication of the genome. Previous research in lower eukaryotes has demonstrated a requisite role for the CDC7 protein kinase in DNA replication origin firing through phosphorylation of the MCM2-7 helicase complex. Owing to its overexpression in various neoplasms, CDC7 has emerged as an attractive target for cancer treatment. We previously reported that SRA141, a potent and selective CDC7 inhibitor, is cytotoxic to multiple tumor cell lines in vitro and demonstrates robust anti-tumor efficacy in colorectal and leukemia xenograft models. Here, we explored the effects of SRA141 on DNA replication and cell cycle dynamics and how these effects relate to the mechanism of SRA141-induced cell death in colorectal cancer cell lines. SRA141 treatment caused a complete inhibition of MCM2 phosphorylation and reduced overall cellular DNA synthesis within 3 hours, with the greatest effects observed in late S-phase. Unexpectedly, DNA combing experiments indicated that SRA141 caused a 70-90% increase in the rate of replication fork progression. These findings suggest that SRA141 may indeed limit origin firing, but this effect is compensated for in part by increased replication fork rates. Despite delay, nucleotide analog pulse-chase experiments indicated that SRA141 treated cells ultimately finished S phase and transitioned into mitosis. In contrast to other reports, no evidence of replication fork collapse was observed, indicating that the main mechanism of SRA141 cytotoxicity is not mediated through replication fork collapse. Intriguingly, some of the most pronounced effects of SRA141 treatment were on progression through M phase, as evidenced by an accumulation of mitotic cells and increased Cyclin B levels following treatment. Moreover, inhibition of Aurora B kinase, which regulates mitotic progression, strongly synergized with SRA141 in caspase-dependent cancer cell killing. Taken together, our findings indicate that the mechanism of cytotoxicity of CDC7 inhibitors is distinct from agents that cause replication fork collapse or inhibit CDKs, and thus may define a new class of cancer therapeutic agents that synergize with drugs that target certain mitotic pathways. Citation Format: Veena Jagannathan, Snezana Milutinovic, Ryan Hansen, Bryan Strouse, Christian Hassig, Eric Brown. CDC7 kinase inhibition by SRA141 induces a potentially novel caspase-dependent tumor cell apoptosis associated with altered DNA replication and cell cycle dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-288.

Ribosome clearance during RNA interference.

In the course of identifying and cleaving RNA, the RNAi machinery must encounter and contend with the megadalton-sized ribosomes that carry out translation. We investigated this interface by examining the fate of actively translated mRNAs subjected to RNAi in C. elegans. Quantifying RNA levels (RNA-seq) and ongoing translation (Ribo-seq), we found there is a greater fold repression of ongoing translation than expected from loss of RNA alone, observing stronger translation repression relative to RNA repression for multiple, independent double-stranded RNA triggers, and for multiple genes. In animals that lack the RNA helicase SKI complex and the ribosome rescue factor PELOTA, ribosomes stall on the 3′ edges of mRNAs at and upstream of the RNAi trigger. One model to explain these observations is that ribosomes are actively cleared from mRNAs by SKI and PELO during or following mRNA cleavage. Our results expand prior studies that show a role for the SKI RNA helicase complex in removing RNA targets following RNAi in flies and plants, illuminating the widespread role of the nonstop translation surveillance in RNA silencing during RNAi. Our results are also consistent with proposals that RNAi can attack messages during active translation.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

Evolutionary Rate Correlation between Mitochondrial-Encoded and Mitochondria-Associated Nuclear-Encoded Proteins in Insects.

The mitochondrion is a pivotal organelle for energy production, and includes components encoded by both the mitochondrial and nuclear genomes. Functional and evolutionary interactions are expected between the nuclear- and mitochondrial-encoded components. The topic is of broad interest in biology, with implications to genetics, evolution, and medicine. Here, we compare the evolutionary rates of mitochondrial proteins and ribosomal RNAs to rates of mitochondria-associated nuclear-encoded proteins, across the major orders of holometabolous insects. There are significant evolutionary rate correlations (ERCs) between mitochondrial-encoded and mitochondria-associated nuclear-encoded proteins, which are likely driven by different rates of mitochondrial sequence evolution and correlated changes in the interacting nuclear-encoded proteins. The pattern holds after correction for phylogenetic relationships and considering protein conservation levels. Correlations are stronger for both nuclear-encoded OXPHOS proteins that are in contact with mitochondrial OXPHOS proteins and for nuclear-encoded mitochondrial ribosomal amino acids directly contacting the mitochondrial rRNAs. We find that ERC between mitochondrial- and nuclear-encoded proteins is a strong predictor of nuclear-encoded proteins known to interact with mitochondria, and ERC shows promise for identifying new candidate proteins with mitochondrial function. Twenty-three additional candidate nuclear-encoded proteins warrant further study for mitochondrial function based on this approach, including proteins in the minichromosome maintenance helicase complex.

HERC2 Facilitates BLM and WRN Helicase Complex Interaction with RPA to Suppress G-Quadruplex DNA.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg Cancer Res; 78(22); 6371-85. ©2018 AACR.

Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP

Centromeric chromatin defines the site of kinetochore formation and ensures faithful chromosome segregation. Centromeric identity is epigenetically specified by the incorporation of CENP-A nucleosomes. DNA replication presents a challenge for inheritance of centromeric identity because nucleosomes are removed to allow for replication fork progression. Despite this challenge, CENP-A nucleosomes are stably retained through S phase. We used BioID to identify proteins transiently associated with CENP-A during DNA replication. We found that during S phase, HJURP transiently associates with centromeres and binds to pre-existing CENP-A, suggesting a distinct role for HJURP in CENP-A retention. We demonstrate that HJURP is required for centromeric nucleosome inheritance during Sphase. HJURP co-purifies with the MCM2-7 helicase complex and, together with the MCM2 subunit, binds CENP-A simultaneously. Therefore, pre-existing CENP-A nucleosomes require an S phase function of the HJURP chaperone and interaction with MCM2 to ensure faithful inheritance of centromere identity through DNA replication.

Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier–Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1–MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.