Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
J Biol Chem ; 300(1): 105503, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38013090

RESUMEN

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Thermococcus , Desoxirribonucleasa IV (Fago T4-Inducido) , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Thermococcus/enzimología , Thermococcus/genética
2.
Appl Environ Microbiol ; 88(2): e0213721, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-34788065

RESUMEN

It has been predicted that 30 to 80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing an ∼5-fold average coverage of the T. kodakarensis genome were prepared in Escherichia coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') that is found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing, and capillary electrophoresis. The method described here provides the details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next-generation sequencing, and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities, including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.


Asunto(s)
Proteínas Arqueales , Thermococcus , Proteínas Arqueales/metabolismo , ADN de Archaea/genética , ADN de Archaea/metabolismo , Electroforesis Capilar , Escherichia coli/genética , Escherichia coli/metabolismo , Genómica
3.
Biochemistry ; 60(3): 210-218, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33426868

RESUMEN

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (εA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to εA excision by utilizing a global analysis of 21 εA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of εA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN/química , Histonas/química , Nucleosomas/química , Proteínas de Xenopus/química , Acetilación , Animales , ADN Glicosilasas/química , Xenopus laevis
4.
Chem Res Toxicol ; 33(7): 1888-1896, 2020 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-32293880

RESUMEN

1,N6-ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145-147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on εA lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of εA at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of εA and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded εA lesions, but only AAG was capable of full repair of any εA in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.


Asunto(s)
Adenina/análogos & derivados , Reparación del ADN , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/química , ADN/química , ADN Glicosilasas/química , Modelos Moleculares , Nucleosomas
5.
Mutagenesis ; 35(1): 39-50, 2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-31612219

RESUMEN

DNA is comprised of chemically reactive nucleobases that exist under a constant barrage from damaging agents. Failure to repair chemical modifications to these nucleobases can result in mutations that can cause various diseases, including cancer. Fortunately, the base excision repair (BER) pathway can repair modified nucleobases and prevent these deleterious mutations. However, this pathway can be hindered through several mechanisms. For instance, mutations to the enzymes in the BER pathway have been identified in cancers. Biochemical characterisation of these mutants has elucidated various mechanisms that inhibit their activity. Furthermore, the packaging of DNA into chromatin poses another obstacle to the ability of BER enzymes to function properly. Investigations of BER in the base unit of chromatin, the nucleosome core particle (NCP), have revealed that the NCP acts as a complex substrate for BER enzymes. The constituent proteins of the NCP, the histones, also have variants that can further impact the structure of the NCP and may modulate access of enzymes to the packaged DNA. These histone variants have also displayed significant clinical effects both in carcinogenesis and patient prognosis. This review focuses on the underlying molecular mechanisms that present obstacles to BER and the relationship of these obstacles to cancer. In addition, several chemotherapeutics induce DNA damage that can be repaired by the BER pathway and understanding obstacles to BER can inform how resistance and/or sensitivity to these therapies may occur. With the understanding of these molecular mechanisms, current chemotherapeutic treatment regiments may be improved, and future therapies developed.


Asunto(s)
Carcinogénesis/genética , Carcinogénesis/metabolismo , Cromatina/química , Cromatina/metabolismo , Reparación del ADN , ADN/metabolismo , Cromatina/genética , Daño del ADN , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN Ligasas/genética , ADN Ligasas/metabolismo , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Resistencia a Antineoplásicos , Histonas/química , Histonas/metabolismo , Humanos
6.
DNA Repair (Amst) ; 71: 87-92, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30170831

RESUMEN

The base excision repair (BER) pathway removes modified nucleobases that can be deleterious to an organism. BER is initiated by a glycosylase, which finds and removes these modified nucleobases. Most of the characterization of glycosylase activity has been conducted in the context of DNA oligomer substrates. However, DNA within eukaryotic organisms exists in a packaged environment with the basic unit of organization being the nucleosome core particle (NCP). The NCP is a complex substrate for repair in which a variety of factors can influence glycosylase activity. In this Review, we focus on the geometric positioning of modified nucleobases in an NCP and the consequences on glycosylase activity and initiating BER.


Asunto(s)
Cromatina/metabolismo , Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , ADN/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Humanos , Nucleosomas/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA