DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

Replication of [AT/TA]25 Microsatellite Sequences by Human DNA Polymerase δ Holoenzymes Is Dependent on dNTP and RPA Levels.

Fragile sites are unstable genomic regions that are prone to breakage during stressed DNA replication. Several common fragile sites (CFS) contain A+T-rich regions including perfect [AT/TA] microsatellite repeats that may collapse into hairpins when in single-stranded DNA (ssDNA) form and coincide with chromosomal hotspots for breakage and rearrangements. While many factors contribute to CFS instability, evidence exists for replication stalling within [AT/TA] microsatellite repeats. Currently, it is unknown how stress causes replication stalling within [AT/TA] microsatellite repeats. To investigate this, we utilized FRET to characterize the structures of [AT/TA]25 sequences and also reconstituted lagging strand replication to characterize the progression of pol δ holoenzymes through A+T-rich sequences. The results indicate that [AT/TA]25 sequences adopt hairpins that are unwound by the major ssDNA-binding complex, RPA, and the progression of pol δ holoenzymes through A+T-rich sequences saturated with RPA is dependent on the template sequence and dNTP concentration. Importantly, the effects of RPA on the replication of [AT/TA]25 sequences are dependent on dNTP concentration, whereas the effects of RPA on the replication of A+T-rich, nonstructure-forming sequences are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how [AT/TA] microsatellite repeats contribute to genome instability.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

Elucidation of unusual biosynthesis and DnaN-targeting mode of action of potent anti-tuberculosis antibiotics Mycoplanecins.

DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2 S,4 R)-4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from the 4-methylproline pathway. We isolate and structurally elucidate four mycoplanecins comprising scarce homo-amino acids and 4-alkylprolines. Evaluating mycoplanecin E against Mycobacterium tuberculosis surprisingly reveals an excitingly low minimum inhibition concentration at 83 ng/mL, thus outcompeting griselimycin by approximately 24-fold. We show that mycoplanecins bind DnaN with nanomolar affinity and provide a co-crystal structure of mycoplanecin A-bound DnaN. Additionally, we reconstitute the biosyntheses of the unusual L-homoleucine, L-homonorleucine, and (2 S,4 R)-4-ethylproline building blocks by characterizing in vitro the full set of eight enzymes involved. The biosynthetic study, bioactivity evaluation, and drug target validation of mycoplanecins pave the way for their further development to tackle multidrug-resistant mycobacterial infections.

Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease all over the world, and no drug is approved for the treatment of NAFLD. Bavachinin (BVC) is proven to possess liver-protecting effect against NAFLD, but its mechanism is still blurry. OBJECTIVES: With the use of Click Chemistry-Activity-Based Protein Profiling (CC-ABPP) technology, this study aims to identify the target of BVC, and investigate the mechanism by which BVC exerts its liver-protecting effect. METHODS: The high fat diet induced hamster NAFLD model is introduced to investigate BVC's lipid-lowering and liver-protecting effects. Then, a small molecular probe ofBVC is designed and synthesized based on theCC-ABPP technology, and BVC's target is fished out. A series of experiments are performed to identify the target, including competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and co-immunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). RESULT: In the hamster NAFLD model, BVC shows lipid-lowing effect and improvement on the histology. PCNA is identified as the target of BVC with the method mentioned above, and BVC facilitates the interaction between PCNA and DNA polymerase delta. BVC promotes HepG2 cells proliferation which is inhibited by T2AA, an inhibitor suppresses the interaction between PCNA and DNA polymerase delta. In NAFLD hamsters, BVC enhances PCNA expression and liver regeneration, reduces hepatocyte apoptosis. CONCLUSION: This study suggests that, besides the anti-lipemic effect, BVC binds to the pocket of PCNA facilitating its interaction with DNA polymerase delta and pro-regeneration effect, thereby exerts the protective effect against HFD induced liver injury.

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism.

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.

Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

A four-point molecular handover during Okazaki maturation.

DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.

Break-induced replication orchestrates resection-dependent template switching.

Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3-5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.

DNA polymerase ε leading strand signature mutations result from defects in its proofreading activity.

The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.

DNA polymerase δ: A single Pol31 polymorphism suppresses the strain background-specific lethality of Pol32 inactivation in Saccharomyces cerevisiae.

The evolutionarily conserved DNA polymerase delta (Polδ) plays several essential roles in eukaryotic DNA replication and repair, responsible for the synthesis of the lagging-strand, lower replicative mutagenesis via its proof-reading exonuclease activity and synthetizes both strands during break-induced replication. In Saccharomyces cerevisiae, the Polδ protein complex consists of three subunits encoded by the POL3, POL31 and POL32 genes. Surprisingly, in contrast to POL3 and POL31, the POL32 gene deletion was found to be viable but lethal in all other eukaryotes, raising the question to which extent the viability of the POL32 deletion in S. cerevisiae was species specific. To address this issue, we inactivated the POL32 gene in 10 evolutionary close or distant S. cerevisiae strains and found that POL32 was either essential (3 strains including SK1), non-essential (5 strains including the reference S288C strain) or confers a slow-growth phenotype (2 strains). Whole-genome sequencing of S288C/SK1 pol32∆ meiotic segregants identified the lethal/suppressor effect of the single Pol31-C43Y polymorphism. Consistently, the introduction of the Pol31-43C allele in the SK1 and West African (WA) pol32∆ mutants was sufficient to restore cell viability and wild-type growth upon introduction of two copies of POL31-43C in the SK1 haploid strain. Reciprocally, introduction of the SK1 POL31-43Y allele in the S288C pol32∆ mutant was lethal. Sequence analyses of the POL31 polymorphisms in the 1,011 yeasts genome dataset correlates with the strict occurrence of the POL31-43Y allele in the yeast African palm wine clade. Differently, the single Pol31-E400G polymorphism confers pol32∆ lethality in the Malaysian strain. In the yeast two-hybrid assay, we observed a weakened interaction between Pol3 and Pol31-43Y versus Pol31-43C suggesting an insufficient level of the Polδ holoenzyme stability/activity. Thus, the enigmatic non-essentiality of Pol32 in S. cerevisiae results from single Pol31 amino acid polymorphism and is clade rather than species specific.

Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination.

Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis.

DNA polymerase POLD1 promotes proliferation and metastasis of bladder cancer by stabilizing MYC.

To date, most studies on the DNA polymerase, POLD1, have focused on the effect of POLD1 inactivation mutations in tumors. However, the implications of high POLD1 expression in tumorigenesis remains elusive. Here, we determine that POLD1 has a pro-carcinogenic role in bladder cancer (BLCA) and is associated to the malignancy and prognosis of BLCA. Our studies demonstrate that POLD1 promotes the proliferation and metastasis of BLCA via MYC. Mechanistically, POLD1 stabilizes MYC in a manner independent of its' DNA polymerase activity. Instead, POLD1 attenuates FBXW7-mediated ubiquitination degradation of MYC by directly binding to the MYC homology box 1 domain competitively with FBXW7. Moreover, we find that POLD1 forms a complex with MYC to promote the transcriptional activity of MYC. In turn, MYC increases expression of POLD1, forming a POLD1-MYC positive feedback loop to enhance the pro-carcinogenic effect of POLD1-MYC on BLCA. Overall, our study identifies POLD1 as a promotor of BCLA via a MYC driven mechanism and suggest its potential as biomarker for BLCA.

DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation.

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Functional hierarchy of PCNA-interacting motifs in DNA processing enzymes.

Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA-interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo-EM reconstructions of high-fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ, and Lig1.

POLD3 deficiency is associated with severe combined immunodeficiency, neurodevelopmental delay, and hearing impairment.

Combined immunodeficiency diseases (CID) represent the most severe forms of inborn errors of immunity. Defective T cell development and/or function, leading to an impairment in adaptive immunity are responsible for these diseases. The DNA polymerase δ complex is important for genome duplication and maintenance and consists of the catalytic subunit POLD1, and the accessory subunits POLD2 and POLD3 which stabilizes the complex. Mutations in POLD1 and POLD2 have been recently shown to be associated with a syndromic CID characterized by T cell lymphopenia with or without intellectual deficiency and sensorineural hearing loss. Here we report a homozygous POLD3 variant (NM_006591.3; p.Ile10Thr) in a Lebanese patient, the product of a consanguineous family, presenting with a syndromic severe combined immunodeficiency (SCID) with neurodevelopmental delay and hearing loss. The homozygous POLD3Ile10Thr variant abolishes POLD3 as well as POLD1 and POLD2 expression. Our findings implicate POLD3 deficiency as a novel cause of syndromic SCID.

Mismatch repair operates at the replication fork in direct competition with mismatch extension by DNA polymerase δ.

DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase δ (Polδ) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Polδ proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Polδ and Polε. Our finding that suppression of elevated mutagenesis in pol3-01 by the Polδ pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Polδ. Furthermore, the evidence that Polδ pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Polδ in replication of both the leading and lagging DNA strands.