Production, Extraction, and Purification of Human Poly(ADP-ribose) Polymerase-1 (PARP-1) with High Specific Activity

Knight, Matthew I.; Chambers, Paul J.

doi:10.1006/prep.2001.1513

Cited by 10 publications

(5 citation statements)

References 22 publications

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“…This concept originates from the differences in the K M and K cat /K M of both enzymes for NAD + , which indicate that PARP-1 is a faster and more efficient NAD + consumer than SIRT1 (Knight and Chambers, 2001; Smith et al, 2009). Therefore, it is likely that PARP-1 activity maintains NAD + at limiting levels for SIRT1 function.…”

Section: Discussionmentioning

confidence: 99%

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

et al. 2011

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Summary SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD+ levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD+ availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD+-consuming enzyme, increases NAD+ content and SIRT1 activity in brown adipose tissue and muscle. PARP-1−/− mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD+ content, SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that not only could be useful in the management of metabolic diseases but also of cancer.

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Section: Discussionmentioning

confidence: 99%

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

et al. 2011

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“…LigIII was overexpressed in and purified from bacteria as described 65 . PARP-1 was expressed as His-tag protein and purified using Ni-agarose column, followed by a cation exchange 66 . For GST pull-down, the GST alone or GST-FUS purified protein was incubated with PARP-1, XRCC1/LigIII complex or individual XRCC1 and LigIII at 4 °C overnight with EZview Red Glutathione Affinity Gel (Sigma), and beads were then washed for three time with TEN buffer (20 mM Tris⋅HCl, pH 7.4, 0.1 mM EDTA and 100 mM NaCl) and boiled in 4 × SDS loading buffer, and the proteins were analyzed by immunoblotting with the indicated antibodies 62 .…”

Section: Methodsmentioning

confidence: 99%

Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis

et al. 2018

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Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression.

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“…Recombinant PARP-1 was produced in Sf9 cells using the Baculovirus Constructs, pFastBac1-PARP-1 (kindly provided by Dr Matthew Knight) (37). Small-scale stocks of baculovirus were prepared from monolayer cultures and large-scale stocks from suspension cultures.…”

Section: Methodsmentioning

confidence: 99%

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

Rosidi¹,

Wang²,

Sharma³

et al. 2008

Nucleic Acids Research

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DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

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Production, Extraction, and Purification of Human Poly(ADP-ribose) Polymerase-1 (PARP-1) with High Specific Activity

Cited by 10 publications

References 22 publications

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis

Histone H1 functions as a stimulatory factor in backup pathways of NHEJ

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