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1.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
2.
Genetics ; 208(1): 125-138, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29162625

RESUMO

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.


Assuntos
Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Dano ao DNA , Ativação Enzimática , Exodesoxirribonucleases/deficiência , Instabilidade Genômica , Recombinação Homóloga , Mutação , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , RecQ Helicases/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
Nucleic Acids Res ; 45(20): 11878-11890, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29059328

RESUMO

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.


Assuntos
DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , RecQ Helicases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Magnetismo , Modelos Moleculares , Conformação de Ácido Nucleico , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , RecQ Helicases/química
4.
Structure ; 25(10): 1582-1588.e3, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28919440

RESUMO

Topoisomerase IIß binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/química , RecQ Helicases/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Conformação Proteica , Serina/metabolismo , Transativadores/metabolismo
5.
J Biol Chem ; 292(35): 14576-14586, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28679532

RESUMO

Double strand breaks (DSBs) represent highly deleterious DNA damage and need to be accurately repaired. Homology-directed repair and non-homologous end joining (NHEJ) are the two major DSB repair pathways that are highly conserved from yeast to mammals. The choice between these pathways is largely based on 5' to 3' DNA resection, and NHEJ proceeds only if resection has not been initiated. In yeast, yKu70/80 rapidly localizes to the break, protecting DNA ends from nuclease accessibility, and recruits additional NHEJ factors, including Nej1 and Lif1. Cells harboring the nej1-V338A mutant exhibit NHEJ-mediated repair deficiencies and hyper-resection 0.15 kb from the DSB that was dependent on the nuclease activity of Dna2-Sgs1. The integrity of Nej1 is also important for inhibiting long-range resection, 4.8 kb from the break, and for preventing the formation of large genomic deletions at sizes >700 bp around the break. Nej1V338A localized to a DSB similarly to WT Nej1, indicating that the Nej1-Lif1 interaction becomes critical for blocking hyper-resection mainly after their recruitment to the DSB. This work highlights that Nej1 inhibits 5' DNA hyper-resection mediated by Dna2-Sgs1, a function distinct from its previously reported role in supporting Dnl4 ligase activity, and has implications for repair pathway choice and resection regulation upon DSB formation.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Deleção de Genes , Viabilidade Microbiana , Mutação Puntual , Multimerização Proteica , Transporte Proteico , RecQ Helicases/química , RecQ Helicases/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie
6.
Biochem Biophys Res Commun ; 486(4): 1116-1121, 2017 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-28385527

RESUMO

Mutations in the human RecQ4 DNA helicase are associated with three different diseases characterized by genomic instability. To gain insight into how RecQ4 dysfunction leads to these pathologies, several groups have used the Saccharomyces cerevisiae RecQ4 homolog Hrq1 as an experimental model. Hrq1 displays many of the same functions as RecQ4 in vivo and in vitro. However, there is some disagreement in the literature about the effects of single-stranded DNA (ssDNA) length on Hrq1 helicase activity and the ability of Hrq1 to anneal complementary ssDNA oligonucleotides into duplex DNA. Here, we present a side-by-side comparison of Hrq1 and RecQ4 helicase activity, demonstrating that in both cases, long random-sequence 3' ssDNA tails inhibit DNA unwinding in vitro in a length-dependent manner. This appears to be due to the formation of secondary structures in the random-sequence ssDNA because Hrq1 preferentially unwound poly(dT)-tailed forks independent of ssDNA length. Further, RecQ4 is capable of ssDNA strand annealing and annealing-dependent strand exchange, but Hrq1 lacks these activities. These results establish the importance of DNA sequence in Hrq1 helicase activity, and the absence of Hrq1 strand annealing activity explains the previously identified discrepancies between S. cerevisiae Hrq1 and human RecQ4.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , RecQ Helicases/química , RecQ Helicases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Bases , Sítios de Ligação , Ativação Enzimática/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Relação Estrutura-Atividade
7.
Nucleic Acids Res ; 45(9): 5217-5230, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28334827

RESUMO

The five human RecQ helicases participate in multiple processes required to maintain genome integrity. Of these, the disease-linked RecQ4 is the least studied because it poses many technical challenges. We previously demonstrated that the yeast Hrq1 helicase displays similar functions to RecQ4 in vivo, and here, we report the biochemical and structural characterization of these enzymes. In vitro, Hrq1 and RecQ4 are DNA-stimulated ATPases and robust helicases. Further, these activities were sensitive to DNA sequence and structure, with the helicases preferentially unwinding D-loops. Consistent with their roles at telomeres, telomeric repeat sequence DNA also stimulated binding and unwinding by these enzymes. Finally, electron microscopy revealed that Hrq1 and RecQ4 share similar structural features. These results solidify Hrq1 as a true RecQ4 homolog and position it as the premier model to determine how RecQ4 mutations lead to genomic instability and disease.


Assuntos
Doença/genética , RecQ Helicases/química , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia Estrutural de Proteína , DNA/metabolismo , Reparo do DNA , Vetores Genéticos/metabolismo , Humanos , Cinética , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Especificidade por Substrato , Telômero/genética
8.
J Biol Chem ; 292(14): 5909-5920, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28228481

RESUMO

Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a defined three-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Galinhas , Multimerização Proteica , RecQ Helicases/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Cristalografia por Raios X , Domínios Proteicos , Estrutura Quaternária de Proteína , RecQ Helicases/genética , RecQ Helicases/metabolismo
9.
J Biol Chem ; 292(10): 4313-4325, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28159839

RESUMO

Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.


Assuntos
Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , RecQ Helicases/metabolismo , Proteínas Ribossômicas/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Células Cultivadas , Dano ao DNA , Reparo do DNA , Replicação do DNA , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunoprecipitação , Mutagênese Sítio-Dirigida , Mutação/genética , Osteossarcoma/genética , Osteossarcoma/patologia , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética
10.
Proc Natl Acad Sci U S A ; 114(4): E466-E475, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28069956

RESUMO

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.


Assuntos
DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinação Homóloga , RecQ Helicases/metabolismo , Reparo do DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Sequências Repetidas Invertidas , RecQ Helicases/química
11.
Nucleic Acids Res ; 45(7): 4231-4243, 2017 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-28100692

RESUMO

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.


Assuntos
Modelos Moleculares , RecQ Helicases/química , Domínio Catalítico , Humanos , Mutação , Conformação Proteica em alfa-Hélice , RecQ Helicases/genética , RecQ Helicases/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
12.
J Biol Chem ; 292(10): 4176-4184, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-27998982

RESUMO

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix ß-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.


Assuntos
DNA/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , RecQ Helicases/química , RecQ Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , Biologia Computacional , Cristalografia por Raios X , DNA/química , DNA/genética , Bases de Dados de Proteínas , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação/genética , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RecQ Helicases/genética
13.
Proc Natl Acad Sci U S A ; 113(30): 8448-53, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27407146

RESUMO

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.


Assuntos
RNA Helicases DEAD-box/química , DNA/química , Quadruplex G , RecQ Helicases/química , Imagem Individual de Molécula/métodos , Helicase da Síndrome de Werner/química , Sequência de Bases , Dicroísmo Circular , RNA Helicases DEAD-box/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RecQ Helicases/metabolismo , Especificidade por Substrato , Telômero/genética , Telômero/metabolismo , Helicase da Síndrome de Werner/metabolismo
14.
Mutat Res ; 790: 8-18, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27248010

RESUMO

RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQ1 contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology.


Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Mutagênese Sítio-Dirigida , Mutação Puntual , Domínios Proteicos/genética , RecQ Helicases/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Sequência Conservada , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , RecQ Helicases/química , Alinhamento de Sequência
15.
Nat Commun ; 7: 11242, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27063109

RESUMO

The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ.


Assuntos
Antígenos Nucleares/metabolismo , Sequência Conservada , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Humanos , Autoantígeno Ku , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RecQ Helicases/química , RecQ Helicases/metabolismo , Helicase da Síndrome de Werner
16.
Oncotarget ; 7(22): 32351-61, 2016 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-27083049

RESUMO

Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.


Assuntos
Síndrome de Bloom/enzimologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Anemia de Fanconi/enzimologia , Neoplasias/enzimologia , RecQ Helicases/metabolismo , Transdução de Sinais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Reagentes para Ligações Cruzadas/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Osteossarcoma/enzimologia , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RecQ Helicases/química , RecQ Helicases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Transfecção , Raios Ultravioleta
17.
Nucleic Acids Res ; 44(5): 1989-2006, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26883636

RESUMO

Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as 'knots' within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA Helicases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Quadruplex G , RecQ Helicases/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , DNA Helicases/química , DNA Helicases/genética , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Expressão Gênica , Guanina/química , Guanina/metabolismo , Humanos , RecQ Helicases/química , RecQ Helicases/genética , Helicase da Síndrome de Werner
18.
Sci Rep ; 6: 21501, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26888063

RESUMO

The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability.


Assuntos
RecQ Helicases/química , Proteínas de Xenopus/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis
19.
Biochim Biophys Acta ; 1864(5): 594-608, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26917473

RESUMO

Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.


Assuntos
Malária Falciparum/enzimologia , Plasmodium falciparum/genética , RecQ Helicases/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , DNA de Protozoário/genética , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , RNA de Cadeia Dupla/genética , RecQ Helicases/química , RecQ Helicases/metabolismo
20.
Proc Natl Acad Sci U S A ; 112(50): E6852-61, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26540728

RESUMO

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.


Assuntos
DNA Viral/química , Conformação de Ácido Nucleico , RecQ Helicases/metabolismo , Bacteriófago lambda/genética , Fluorescência , Corantes Fluorescentes/química , Microscopia/métodos , RecQ Helicases/química
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