Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β.
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ABSTRACT:
SUBMITTER: Yuhas SC
PROVIDER: S-EPMC8373715 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.
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Project description:DNA polymerase β (pol β) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol β lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol β, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol β within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol β catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol β kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.
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Project description:Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.
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Project description:DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of gamma-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase beta (Pol beta), an integral enzyme in base-excision repair. Irreversible inhibition of Pol beta by DOB suggests that this lesion provides a chemical explanation for the cytotoxicity of drugs that produce it and explains previously unexplained observations in the literature concerning abasic lesions that are not repaired efficiently. Finally, these observations provide the impetus for the design of a new family of inhibitors of Pol beta.
| S-EPMC2863033 | biostudies-literature
Project description:DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol β scans DNA using a processive hopping mechanism and has a mean search footprint of ∼24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol β. Application of our processive search assay into nucleosome core particles revealed that Pol β is not processive in the context of a nucleosome, and its single-turnover activity is reduced ∼500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol β mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol β.
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Project description:The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 β-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation. These results suggest new principles of covalent drug design and off-target site prediction. Unlike shape and electrostatic complementary which address binding constants, electrostatic catalysis drives reaction rates, essential for covalent inhibition, and deepens our understanding of chemical reactivity, selectivity, and stability in complex systems.
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Project description:PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase ? (Pol ?). Its functions in DNA replication are unknown. PDIP46 associates with Pol ? in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol ? activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol ?. The effects of PDIP46 on Pol ? in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol ?4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol ?, independent of PCNA. Mutation of the Pol ? and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol ? that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol ? functions in vivo, and thereby be a nexus for altered genomic stability.
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Project description:We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.
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Project description:The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase ? when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase ? was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase ? and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase ? variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.
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Project description:DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (kpol), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.
| S-EPMC8277322 | biostudies-literature