Pol Domain Research Articles

Abstract 3104: A NanoBRET target engagement assay for querying domain selectivity at full-length polymerase theta in live cells

Abstract In HR-deficient cells, Polymerase Theta (Pol-theta) serves to repair double strand breaks and has emerged as a therapeutic target in HR-deficient cancers. Encoded by the POLQ gene, Pol-theta is a large (290 Kda) protein with two distinct functional domains (polymerase, helicase), separated by an unstructured region. Each domain of Pol-theta has proven vulnerable to small molecule inhibitors, with a number of molecules undergoing clinical evaluation. Unlike the helicase domain, target engagement at the polymerase domain requires cooperativity with DNA fragments. Here we describe a novel NanoBRET Target Engagement assay that enables domain-specific analysis of small molecule occupancy in live cells. To observe the high affinity-state of small molecule inhibitors to the polymerase domain, we have developed a cell-permeable pol domain NanoBRET probe, along with a novel work flow to introduce small DNA fragments into live cells. Together these components form a nucleoprotein complex with full length polymerase theta. This assay method can be used to quantify and rank-order engagement to the polymerase domain in live cells. To enable domain-selectivity analysis, we have developed a second NanoBRET probe directed to the helicase domain. Together these two NanoBRET probes allow for the mechanism of action studies at Pol-theta in living cells. Citation Format: Steven Edenson, Michael Slater, Cesear Corona, Michael Beck, James Vasta, Kelly Teske, Ani Michaud, Matthew Robers. A NanoBRET target engagement assay for querying domain selectivity at full-length polymerase theta in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3104.

Reclassification of family A DNA polymerases reveals novel functional subfamilies and distinctive structural features.

Family A DNA polymerases (PolAs) form an important and well-studied class of extant polymerases participating in DNA replication and repair. Nonetheless, despite the characterization of multiple subfamilies in independent, dedicated works, their comprehensive classification thus far is missing. We therefore re-examine all presently available PolA sequences, converting their pairwise similarities into positions in Euclidean space, separating them into 19 major clusters. While 11 of them correspond to known subfamilies, eight had not been characterized before. For every group, we compile their general characteristics, examine their phylogenetic relationships and perform conservation analysis in the essential sequence motifs. While most subfamilies are linked to a particular domain of life (including phages), one subfamily appears in Bacteria, Archaea and Eukaryota. We also show that two new bacterial subfamilies contain functional enzymes. We use AlphaFold2 to generate high-confidence prediction models for all clusters lacking an experimentally determined structure. We identify new, conserved features involving structural alterations, ordered insertions and an apparent structural incorporation of a uracil-DNA glycosylase (UDG) domain. Finally, genetic and structural analyses of a subset of T7-like phages indicate a splitting of the 3'-5' exo and pol domains into two separate genes, observed in PolAs for the first time.

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

Cyclin‐dependent kinase 12‐mediated super‐enhancers as promising targets in cancer therapy

CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation

CDK9 is a kinase critical for the productive transcription of protein‐coding genes by RNA polymerase II (pol II). As part of P‐TEFb, CDK9 phosphorylates the carboxyl‐terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein‐coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation.

The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

RETRACTED: Human DNA polymerase θ harbors DNA end-trimming activity critical for DNA repair

Cancers with hereditary defects in homologous recombination rely on DNA polymerase θ (pol θ) for repair of DNA double-strand breaks. During end joining, pol θ aligns microhomology tracts internal to 5'-resected broken ends. An unidentified nuclease trims the 3' ends before synthesis can occur. Here we report that a nuclease activity, which differs from the proofreading activity often associated with DNA polymerases, is intrinsic to the polymerase domain of pol θ. Like the DNA synthesis activity, the nuclease activity requires conserved metal-binding residues, metal ions, and dNTPs and is inhibited by ddNTPs or chain-terminated DNA. Our data indicate that pol θ repurposes metal ions in the polymerase active site for endonucleolytic cleavage and that the polymerase-active and end-trimming conformations of the enzyme are distinct. We reveal a nimble strategy of substrate processing that allows pol θ to trim or extend DNA depending on the DNA repair context.

Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

A multifunctional DNA polymerase I involves in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in Helicobacterpylori.

Helicobacterpylori is the most infectious human pathogen that causes gastritis, peptic ulcers and stomach cancer. H.pylori DNA polymerase I (HpPol I) is found to be essential for the viability of H.pylori, but its intrinsic property and attribution to the H.pylori DNA replication remain unclear. HpPol I contains a 5'→3' exonuclease (5'-Exo) and DNA polymerase (Pol) domain, respectively, but lacks a 3'→5' exonuclease, or error proofreading activity. In this study, we characterized the 5'-Exo and Pol functions of HpPol I and found that HpPol I is a multifunctional protein displaying DNA nick translation, strand-displacement synthesis, RNase H-like, structure-specific endonuclease and exonuclease activities. In the invitro DNA replication assay, we further demonstrated that the 5'-Exo and Pol domains of HpPol I can cooperate to fill in the DNA gap, remove the unwanted RNA primer from a RNA/DNA hybrid and create a ligatable nick for the DNA ligase A of H.pylori to restore the normal duplex DNA. Altogether, our study suggests that the two catalytic domains of HpPol I may synergistically play an important role in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in H.pylori. Like the functions of DNA polymerase I in Escherichiacoli, HpPol I may involve in both DNA replication and repair in H.pylori.

Long Tandem Arrays of Cassandra Retroelements and Their Role in Genome Dynamics in Plants.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme.

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

Coevolution analysis of amino-acids reveals diversified drug-resistance solutions in viral sequences: a case study of hepatitis B virus.

The study of mutational landscapes of viral proteins is fundamental for the understanding of the mechanisms of cross-resistance to drugs and the design of effective therapeutic strategies based on several drugs. Antiviral therapy with nucleos(t)ide analogues targeting the hepatitis B virus (HBV) polymerase protein (Pol) can inhibit disease progression by suppression of HBV replication and makes it an important case study. In HBV, treatment may fail due to the emergence of drug-resistant mutants. Primary and compensatory mutations have been associated with lamivudine resistance, whereas more complex mutational patterns are responsible for resistance to other HBV antiviral drugs. So far, all known drug-resistance mutations are located in one of the four Pol domains, called reverse transcriptase. We demonstrate that sequence covariation identifies drug-resistance mutations in viral sequences. A new algorithmic strategy, BIS2TreeAnalyzer, is designed to apply the coevolution analysis method BIS2, successfully used in the past on small sets of conserved sequences, to large sets of evolutionary related sequences. When applied to HBV, BIS2TreeAnalyzer highlights diversified viral solutions by discovering thirty-seven positions coevolving with residues known to be associated with drug resistance and located on the four Pol domains. These results suggest a sequential mechanism of emergence for some mutational patterns. They reveal complex combinations of positions involved in HBV drug resistance and contribute with new information to the landscape of HBV evolutionary solutions. The computational approach is general and can be applied to other viral sequences when compensatory mutations are presumed.

Cutting into the Substrate Dominance: Pharmacophore and Structure-Based Approaches toward Inhibiting Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) contains two distinct functional domains: a DNA polymerase (pol) domain and a ribonuclease H (RNase H) domain, both of which are required for viral genome replication. Over the last 3 decades, RT has been at the forefront of HIV drug discovery efforts with numerous nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) approved by the FDA. However, all these RT inhibitors target only the pol function, and inhibitors of RT-associated RNase H have yet to enter the development pipeline, which in itself manifests both the opportunity and challenges of targeting RNase H: if developed, RT RNase H inhibitors would represent a mechanistically novel class of HIV drugs that can be particularly valuable in treating HIV strains resistant to current drugs. The challenges include (1) the difficulty in selectively targeting RT RNase H over RT pol due to their close interplay both spatially and temporally and over HIV-1 integrase strand transfer (INST) activity because of their active site similarities; (2) to a larger extent, the inability of active site inhibitors to confer significant antiviral effect, presumably due to a steep substrate barrier by which the pre-existing substrate prevents access of small molecules to the active site. As a result, previously reported RT RNase H inhibitors typically lacked target specificity and significant antiviral potency. Achieving meaningful antiviral activity via active site targeting likely entails selective and ultrapotent RNase H inhibition to allow small molecules to cut into the dominance of substrates. Based on a pharmacophore model informed by prior work, we designed and redesigned a few metal-chelating chemotypes, such as 2-hydroxyisoquinolinedione (HID), hydroxypyridonecarboxylic acid (HPCA), 3-hydroxypyrimidine-2,4-dione (HPD), and N-hydroxythienopyrimidine-2,4-dione (HTPD). Analogues of these chemotypes generally exhibited improved potency and selectivity inhibiting RT RNase H over the best previous compounds and further validated the pharmacophore model. Extended structure-activity relationship (SAR) on the HPD inhibitor type by mainly altering the linkage generated a few subtypes showing exceptional potency (single-digit nanomolar) and excellent selectivity over the inhibition of RT pol and INST. In parallel, a structure-based approach also allowed us to design a unique double-winged HPD subtype to potently and selectively inhibit RT RNase H and effectively compete against the RNA/DNA substrate. Significantly, all potent HPD subtypes consistently inhibited HIV-1 in the cell culture, suggesting that carefully designed active site RNase H inhibitors with ultrapotency could partially overcome the barrier to antiviral phenotype. Overall, in addition to identifying our own inhibitor types, our medicinal chemistry efforts demonstrated the value of pharmacophore and structure-based approaches in designing active side-directed RNase H inhibitors and could provide a viable path to validating RNase H as a novel antiviral target.

Abstract 3836: Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML

Abstract Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p < 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of < 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate > 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values < 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [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 3836.

The cap-snatching reaction of yeast L-A double-stranded RNA virus is reversible and the catalytic sites on both Gag and the Gag domain of Gag-Pol are active.

The yeast L-A double-stranded RNA virus synthesizes capped transcripts by a unique cap-snatching mechanism in which the m7 Gp moiety of host mRNA (donor) is transferred to the diphosphorylated 5' end of the viral transcript (acceptor). This reaction is activated by viral transcription. Here, we show that cap snatching can be reversible. Because only m7 Gp is transferred during the reaction, the resulting decapped donor, as expected, retained diphosphates at the 5' end. We also found that the 5' terminal nucleotide of the acceptor needs to be G but not A. Interestingly, the A-initiated molecule when equipped with a cap structure (m7 GpppA…) could work as cap donor. Because the majority of host mRNAs in yeast have A after the cap structures at the 5' ends, this finding implies that cap-snatching in vivo is virtually a one-way reaction, in favor of furnishing the viral transcript with a cap. The cap-snatching sites are located on the coat protein Gag and also the Gag domain of Gag-Pol. Here, we demonstrate that both sites are functional, indicating that activation of cap snatching by transcription is not transmitted through the peptide bonding between the Gag and Pol domains of Gag-Pol.

Analysis of retrotransposon abundance, diversity and distribution in holocentric Eleocharis (Cyperaceae) genomes.

Background and AimsLong terminal repeat-retrotransposons (LTR-RTs) comprise a large portion of plant genomes, with massive repeat blocks distributed across the chromosomes. Eleocharis species have holocentric chromosomes, and show a positive correlation between chromosome numbers and the amount of nuclear DNA. To evaluate the role of LTR-RTs in karyotype diversity in members of Eleocharis (subgenus Eleocharis), the occurrence and location of different members of the Copia and Gypsy superfamilies were compared, covering interspecific variations in ploidy levels (considering chromosome numbers), DNA C-values and chromosomal arrangements.MethodsThe DNA C-value was estimated by flow cytometry. Genomes of Eleocharis elegans and E. geniculata were partially sequenced using Illumina MiSeq assemblies, which were a source for searching for conserved proteins of LTR-RTs. POL domains were used for recognition, comparing families and for probe production, considering different families of Copia and Gypsy superfamilies. Probes were obtained by PCR and used in fluorescence in situ hybridization (FISH) against chromosomes of seven Eleocharis species.Key ResultsA positive correlation between ploidy levels and the amount of nuclear DNA was observed, but with significant variations between samples with the same ploidy levels, associated with repetitive DNA fractions. LTR-RTs were abundant in E. elegans and E. geniculata genomes, with a predominance of Copia Sirevirus and Gypsy Athila/Tat clades. FISH using LTR-RT probes exhibited scattered and clustered signals, but with differences in the chromosomal locations of Copia and Gypsy. The diversity in LTR-RT locations suggests that there is no typical chromosomal distribution pattern for retrotransposons in holocentric chromosomes, except the CRM family with signals distributed along chromatids.ConclusionsThese data indicate independent fates for each LTR-RT family, including accumulation between and within chromosomes and genomes. Differential activity and small changes in LTR-RTs suggest a secondary role in nuclear DNA variation, when compared with ploidy changes.

P-A2 Development of anti-Tat compound: MD Simulation of the Tat/Cyclin T1/CDK9 Complex Revealing the Hidden Catalytic Cavity within the CDK9 Molecule Upon Tat Binding

We applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytiactivity as the target.

The 5′ Nuclease Domain of DNA Polymerase I Mediates a Novel DNA Transfer Pathway during Proofreading

The high fidelity of many DNA polymerases is due, in part, to their ability to remove erroneously incorporated nucleotides at the terminus of the growing primer strand. This “proofreading” activity is catalyzed by the 3′-5′ exonuclease (exo) domain in E. coli DNA polymerase I (Pol I). Structural studies have shown that this domain is spatially separated from the 5′-3′ polymerase domain (pol), which is responsible for incorporation of dNTPs by template-directed polymerization. Pol I also contains another spatially distinct domain, the 5′ nuclease (nuc) domain, which is connected to the enzyme core by a flexible amino acid linker. This domain is involved in cleavage of downstream flaps formed during Okazaki fragment maturation and base excision repair. Using a single-molecule FRET system, we resolved three distinct conformations of Pol I-DNA complexes. Experiments with Pol I mutants established that these states correspond to DNA bound at the pol, exo, or nuc domains. Notably, the DNA substrate can transfer among all three domains without dissociation from the enzyme. We determined rate constants for site switching of model DNA substrates containing a correctly paired primer terminus, a terminal mismatch or an internal mismatch. The presence of mismatches accelerated transfer of DNA from the pol domain to the exo domain, as expected during proofreading. Surprisingly, the mismatches also accelerated transfer of DNA along the pol to nuc to exo domain pathway. These observations reveal the existence of a second intramolecular proofreading pathway, in which mismatched DNA first transfers from the pol domain to the nuc domain and then to the exo domain. Our results suggest an unexpected role for the 5′ nuclease domain in the proofreading activity of Pol I.

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1ʹ, H2ʹ, and H3ʹ exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Novel POLG mutations and variable clinical phenotypes in 13 Italian patients.

POLG gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA) replication and repair. Mutations in POLG have been linked to a spectrum of clinical phenotypes, resulting in autosomal recessive or dominant mitochondrial diseases. These mutations have been associated with heterogeneous phenotypes, presenting with varying severity and at different ages of onset, ranging from the neonatal period to late adult life. We screened 13 patients for POLG mutations. All patients underwent a complete neurological examination, and in most of cases, muscle biopsy was performed. We detected 15 different variations in 13 unrelated Italian patients. Two mutations were novel and mapped in the pol domain (p.Thr989dup and p.Ala847Thr) of the enzyme. We also report new cases carrying controversial variations previously described as incompletely penetrant or a variant of unknown significance. Our study increases the range of clinical presentations associated with mutations in POLG gene, underlining some peculiar clinical features, such as PEO associated with corneal edema, and epilepsy, severe neuropathy with achalasia. The addition of two new substitutions, including the second report of an in-frame duplication, to the growing list of defects increases the value of POLG genetic diagnosis in a range of neurological presentations.

Estimation of long terminal repeat element content in the Helicoverpa zea genome from high-throughput sequencing of bacterial artificial chromosome pools.

The lepidopteran pest insect Helicoverpa zea feeds on cultivated corn and cotton across the Americas where control remains challenging owing to the evolution of resistance to chemical and transgenic insecticidal toxins, yet genomic resources remain scarce for this species. A bacterial artificial chromosome (BAC) library having a mean genomic insert size of 145 ± 20 kbp was created from a laboratory strain of H. zea, which provides ∼12.9-fold coverage of a 362.8 ± 8.8 Mbp (0.37 ± 0.09 pg) flow cytometry estimated haploid genome size. Assembly of Illumina HiSeq 2000 reads generated from 14 pools that encompassed all BAC clones resulted in 165 485 genomic contigs (N50 = 3262 bp; 324.6 Mbp total). Long terminal repeat (LTR) protein coding regions annotated from 181 contigs included 30 Ty1/copia, 78 Ty3/gypsy, and 73 BEL/Pao elements, of which 60 (33.1%) encoded all five functional polyprotein (pol) domains. Approximately 14% of LTR elements are distributed non-randomly across pools of BAC clones.