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1.
Proc Natl Acad Sci U S A ; 119(33): e2203203119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35947614

RESUMO

Human cells encode up to 15 DNA polymerases with specialized functions in chromosomal DNA synthesis and damage repair. In contrast, complex DNA viruses, such as those of the herpesviridae family, encode a single B-family DNA polymerase. This disparity raises the possibility that DNA viruses may rely on host polymerases for synthesis through complex DNA geometries. We tested the importance of error-prone Y-family polymerases involved in translesion synthesis (TLS) to human cytomegalovirus (HCMV) infection. We find most Y-family polymerases involved in the nucleotide insertion and bypass of lesions restrict HCMV genome synthesis and replication. In contrast, other TLS polymerases, such as the polymerase ζ complex, which extends past lesions, was required for optimal genome synthesis and replication. Depletion of either the polζ complex or the suite of insertion polymerases demonstrate that TLS polymerases suppress the frequency of viral genome rearrangements, particularly at GC-rich sites and repeat sequences. Moreover, while distinct from HCMV, replication of the related herpes simplex virus type 1 is impacted by host TLS polymerases, suggesting a broader requirement for host polymerases for DNA virus replication. These findings reveal an unexpected role for host DNA polymerases in ensuring viral genome stability.


Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA , DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Viral/genética , Humanos
2.
Methods Enzymol ; 672: 103-123, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934471

RESUMO

Replicative helicase and polymerase form the leading-strand replisome that unwinds parental DNA and performs continuous leading-strand DNA synthesis. Uncoupling of the helicase-polymerase complex results in replication stress, replication errors, and genome instability. Although numerous replisomes from different biological systems have been reconstituted and characterized, structural investigations of the leading-strand replisome complex are hindered by its large size and dynamics. We have determined the first replisome structure on a fork substrate with bacteriophage T7 replisome as a model system. Here, we summarized our protocols to prepare and characterize the coupled T7 replisome complex. Similar methods can potentially be applied for structural investigations of more complicated replisomes.


Assuntos
Bacteriófago T7 , DNA Polimerase Dirigida por DNA , Bacteriófago T4/genética , Bacteriófago T7/genética , DNA/química , DNA Helicases/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/química
3.
Methods Enzymol ; 672: 125-142, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934472

RESUMO

The genome of prokaryotes can be damaged by a variety of endogenous and exogenous factors, including reactive oxygen species, UV exposure, and antibiotics. To better understand these repair processes and the impact they may have on DNA replication, normal genome maintenance processes can be perturbed by removing or editing associated genes and monitoring DNA repair outcomes. In particular, the replisome activities of DNA unwinding by the helicase and DNA synthesis by the polymerase must be tightly coupled to prevent any appreciable single strand DNA (ssDNA) from accumulating and amplifying genomic stress. If decoupled, vulnerable ssDNA would persist, likely leading to double strand breaks (DSBs) or requiring replication restart mechanisms downstream of a stall. In either case, free 3'-OH strands would exist, resulting from ssDNA gaps in the leading strand or complete DSBs. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) can enzymatically label ssDNA ends with bromo-deoxy uridine triphosphate (BrdU) to detect free 3'-OH DNA ends in the E. coli genome. Labeled DNA ends can be detected and quantified using fluorescence microscopy or flow cytometry. This methodology is useful in applications where in situ investigation of DNA damage and repair are of interest, including effects from enzyme mutations or deletions and exposure to various environmental conditions.


Assuntos
DNA de Cadeia Simples , Escherichia coli , DNA , DNA Nucleotidilexotransferase , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , DnaB Helicases/genética , Escherichia coli/metabolismo , Marcação In Situ das Extremidades Cortadas
4.
Methods Enzymol ; 672: 299-315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934481

RESUMO

Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome-lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA/metabolismo , DNA Polimerase III , DNA Polimerase Dirigida por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo
5.
Methods Enzymol ; 672: 75-102, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934486

RESUMO

Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA. However, when a helicase and DNA polymerase are functionally and physically coupled, they catalyze fast and highly processive leading strand DNA synthesis. DNA polymerase has a 3'-5' exonuclease activity, which removes nucleotides misincorporated in the nascent DNA. DNA synthesis kinetics, processivity, and accuracy are governed by the interplay of the helicase, DNA polymerase, and exonuclease activities within the replisome. This chapter describes quantitative biochemical and biophysical methods to study the coupling of these three critical activities during DNA replication. The methods include real-time quantitation of kinetics of DNA unwinding-synthesis by a coupled helicase-DNA polymerase complex, a 2-aminopurine fluorescence-based assay to map the precise positions of helicase and DNA polymerase with respect to the replication fork junction, and a radiometric assay to study the coupling of DNA polymerase, exonuclease, and helicase activities during processive leading strand DNA synthesis. These methods are presented here with bacteriophage T7 replication proteins as an example but can be applied to other systems with appropriate modifications.


Assuntos
DNA Polimerase Dirigida por DNA , Exonucleases , DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo
6.
Int J Mol Sci ; 23(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35955705

RESUMO

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases (POLI) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival (p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival.


Assuntos
DNA Polimerase Dirigida por DNA , Neoplasias Pancreáticas , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Aprendizado de Máquina , Prognóstico
7.
Nat Commun ; 13(1): 4547, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927262

RESUMO

The DNA polymerase theta (Polθ)-mediated end joining (TMEJ) pathway for repair of chromosomal double strand breaks (DSBs) is essential in cells deficient in other DSB repair pathways, including hereditary breast cancers defective in homologous recombination. Strand-break activated poly(ADP) ribose polymerase 1 (PARP1) has been implicated in TMEJ, but the modest specificity of existing TMEJ assays means the extent of effect and the mechanism behind it remain unclear. We describe here a series of TMEJ assays with improved specificity and show ablation of PARP activity reduces TMEJ activity 2-4-fold. The reduction in TMEJ is attributable to a reduction in the 5' to 3' resection of DSB ends that is essential for engagement of this pathway and is compensated by increased repair by the nonhomologous-end joining pathway. This limited role for PARP activity in TMEJ helps better rationalize the combined employment of inhibitors of PARP and Polθ in cancer therapy.


Assuntos
Poli(ADP-Ribose) Polimerases , Ribose , Difosfato de Adenosina , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA Polimerase Dirigida por DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo
8.
Acta Biochim Biophys Sin (Shanghai) ; 54(5): 637-646, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35920197

RESUMO

Apurinic/apyrimidic (AP) sites are severe DNA damages and strongly block DNA extension by major DNA polymerases. Y-family DNA polymerases possess a strong ability to bypass AP sites and continue the DNA synthesis reaction, which is called translesion synthesis (TLS) activity. To investigate the effect of the molecular structure of the AP site on the TLS efficiency of Dbh, a Y-family DNA polymerase from Sulfolobus acidocaldarius, a series of different AP site analogues (various spacers) are used to characterize the bypass efficiency. We find that not only the molecular structure and atomic composition but also the number and position of AP site analogues determine the TLS efficiency of Dbh. Increasing the spacer length decreases TLS activity. The TLS efficiency also decreases when more than one spacer exists on the DNA template. The position of the AP site analogues is also an important factor for TLS. When the spacer is opposite to the first incorporated dNTPs, the TLS efficiency is the lowest, suggesting that AP sites are largely harmful for the formation of hydrogen bonds. These results deepen our understanding of the TLS activity of Y-family DNA polymerases and provide a biochemical basis for elucidating the TLS mechanism in Sulfolobus acidocaldarius cells.


Assuntos
Sulfolobus acidocaldarius , DNA/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/metabolismo
9.
Nature ; 607(7920): 799-807, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35859169

RESUMO

The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer1-3. However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms of APOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time4. Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss of the translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures.


Assuntos
Desaminases APOBEC , Mutagênese , Neoplasias , Desaminases APOBEC/deficiência , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/metabolismo , Deleção de Genes , Genoma Humano , Humanos , Mutagênese/genética , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Uracila-DNA Glicosidase/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(28): e2204511119, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35867748

RESUMO

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.


Assuntos
Proteínas Cromossômicas não Histona , DNA Polimerase Dirigida por DNA , Vírus da Hepatite A , Hepatite A , Proteínas Intrinsicamente Desordenadas , RNA Nucleotidiltransferases , RNA Viral , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Hepatite A/tratamento farmacológico , Hepatite A/metabolismo , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Camundongos Mutantes , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Replicação Viral/efeitos dos fármacos
11.
Viruses ; 14(8)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35893688

RESUMO

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been the primary interest among studies on antiviral discovery, viral replication kinetics, drug resistance, and viral evolution. Following infection and entry into target cells, the HIV-1 core disassembles, and the viral RT concomitantly converts the viral RNA into double-stranded proviral DNA, which is integrated into the host genome. The successful completion of the viral life cycle highly depends on the enzymatic DNA polymerase activity of RT. Furthermore, HIV-1 RT has long been known as an error-prone DNA polymerase due to its lack of proofreading exonuclease properties. Indeed, the low fidelity of HIV-1 RT has been considered as one of the key factors in the uniquely high rate of mutagenesis of HIV-1, which leads to efficient viral escape from immune and therapeutic antiviral selective pressures. Interestingly, a series of studies on the replication kinetics of HIV-1 in non-dividing myeloid cells and myeloid specific host restriction factor, SAM domain, and HD domain-containing protein, SAMHD1, suggest that the myeloid cell tropism and high rate of mutagenesis of HIV-1 are mechanistically connected. Here, we review not only HIV-1 RT as a key antiviral target, but also potential evolutionary and mechanistic crosstalk among the unique enzymatic features of HIV-1 RT, the replication kinetics of HIV-1, cell tropism, viral genetic mutation, and host SAMHD1 protein.


Assuntos
Transcriptase Reversa do HIV , HIV-1 , Proteína 1 com Domínio SAM e Domínio HD , Tropismo Viral , Antivirais/farmacologia , DNA Polimerase Dirigida por DNA/genética , Genômica , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Mutagênese , Células Mieloides/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Replicação Viral
12.
Bioorg Chem ; 127: 105987, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35777231

RESUMO

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.


Assuntos
DNA Polimerase Dirigida por DNA , Nucleosídeos , DNA Nucleotidilexotransferase/química , DNA Polimerase Dirigida por DNA/química , Humanos , Nucleosídeos/química , Nucleotídeos/química , Timidina
13.
Vet Microbiol ; 272: 109517, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35908441

RESUMO

Pseudorabies virus (PRV) is a ubiquitous and economically important swine alphaherpesvirus that causes devastating swine diseases worldwide. PRV-encoded DNA-dependent DNA polymerase, comprised of the catalytic subunit UL30 and the accessory subunit UL42, is essential for viral replication. PRV UL30 and UL42 act as a heterodimer with UL30 harboring inherent DNA polymerase activity and UL42 conferring processivity on the DNA polymerase holoenzyme. The formation of PRV UL30/UL42 heterodimer holoenzyme through protein-protein interactions is indispensable for viral replication. In work described here, we defined the key domains that mediate PRV UL30/UL42 interaction, and found that the 41 carboxy-terminal amino acids region of PRV UL30 is critical for its interaction with UL42. Intriguingly, a synthetic peptide corresponding to these 41 carboxy-terminal amino acid residues efficiently disrupted PRV UL30/UL42 interaction through competitively binding to UL42. These findings suggest that the peptides from the PRV DNA polymerase UL30/UL42 subunit interface may represent potential targets for designing a novel intervention strategy against PRV infection. This work further strengthens the concept that the herpesvirus DNA polymerase catalytic subunits utilize their extreme carboxy-terminal domains as a conserved mechanism to associate with their cognate accessory subunits, providing us the opportunity of designing novel antiviral agents against herpesvirus infection through disruption of the herpesvirus DNA polymerase subunit interactions.


Assuntos
Herpesvirus Suídeo 1 , Pseudorraiva , Doenças dos Suínos , Animais , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Peptídeos/genética , Peptídeos/farmacologia , Suínos , Replicação Viral
14.
Nucleic Acids Res ; 50(13): 7420-7435, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35819193

RESUMO

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.


Assuntos
Reparo do DNA , Neoplasias , Animais , Cisplatino/uso terapêutico , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Medicina de Precisão , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitinação
15.
Life Sci Alliance ; 5(12)2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35905994

RESUMO

DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases.


Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo
16.
Int J Mol Sci ; 23(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35887293

RESUMO

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA- mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol-1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.


Assuntos
Bacteriófagos , Thermus thermophilus , Sequência de Aminoácidos , Bacteriófagos/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfodiesterase I/metabolismo , Thermus thermophilus/metabolismo
17.
ACS Chem Biol ; 17(7): 1924-1936, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35776893

RESUMO

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O-P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH2-ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3'-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA─NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.


Assuntos
DNA Polimerase Dirigida por DNA , DNA , Domínio Catalítico , DNA/química , Replicação do DNA , DNA Arqueal , DNA Polimerase Dirigida por DNA/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-35649682

RESUMO

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.


Assuntos
Antineoplásicos , Xeroderma Pigmentoso , Antineoplásicos/farmacologia , DNA , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Temozolomida/farmacologia , Xeroderma Pigmentoso/genética
19.
ACS Appl Mater Interfaces ; 14(26): 29483-29490, 2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35700238

RESUMO

A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤103 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from Thermococcus kodakaraensis (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.


Assuntos
Enzimas Imobilizadas , Dióxido de Silício , DNA , DNA Polimerase Dirigida por DNA/química , Enzimas Imobilizadas/genética , Ácidos Nucleicos Imobilizados/química , Reação em Cadeia da Polimerase/métodos , Dióxido de Silício/química
20.
Oncogene ; 41(32): 3969-3977, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35768547

RESUMO

HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.


Assuntos
Neoplasias de Mama Triplo Negativas , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica/genética , Humanos , Nucleotidiltransferases/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
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