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Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Buelow B, Uzunparmak B, Paddock M, Scharenberg AM - PLoS ONE (2009)

Bottom Line: Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR).To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain.Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

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PARP-1 synthesizes long, branched chains of poly-mADPR (PAR) on a wide variety of acceptor proteins in the nucleus, notably PARP-1 itself (which appears to accumulate roughly 90% of cellular PAR).Subsequently, PAR is degraded by Poly-ADP-Ribose-Glycosylase (PARG) into mADPR, which is assumed to diffuse out of the nucleus and into the cytosol. There it can bind to the ion channel TRPM2, leading to the influx of cations, including calcium. Additionally, PARP-1 mediates the recruitment of DNA repair proteins to sites of DNA damage through protein-protein interactions (notably through its BRCT domain) and PARylation. The relationship between PARP-mediated mADPR degradation and DNA damage repair remains unclear.
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pone-0006339-g001: PARP-1 synthesizes long, branched chains of poly-mADPR (PAR) on a wide variety of acceptor proteins in the nucleus, notably PARP-1 itself (which appears to accumulate roughly 90% of cellular PAR).Subsequently, PAR is degraded by Poly-ADP-Ribose-Glycosylase (PARG) into mADPR, which is assumed to diffuse out of the nucleus and into the cytosol. There it can bind to the ion channel TRPM2, leading to the influx of cations, including calcium. Additionally, PARP-1 mediates the recruitment of DNA repair proteins to sites of DNA damage through protein-protein interactions (notably through its BRCT domain) and PARylation. The relationship between PARP-mediated mADPR degradation and DNA damage repair remains unclear.

Mentions: Much less is known about the biochemical mechanisms of PARP-1 activation in the context of O/N stress induced formation of mADPR. Compelling evidence indicates that PARP-1-dependent mADPR formation results in mADPR-mediated activation of the TRPM2 Ca2+ channel (Figure 1 and [21]–[24]). However, there are no data addressing the biochemical context in which PARP-1 activation leads to mADPR formation, or the relationship between these mechanisms and PARP-1′s involvement in the DNA damage response. To better define the biochemistry of PARP-dependent mADPR formation, we reconstituted PARP-1 deficient DT40 cells with either WT or various mutant forms of PARP-1 (Figure 2), and determined the capacity of each mutant to support two correlates of O/N stress-induced mADPR formation: NAD degradation and TRPM2 activation. Our results suggest that catalytic activity, DNA binding, and an intact auto-PARylation domain are required for PARP-1-mediated cytosolic mADPR accumulation in vivo. In contrast, the BRCT domain, which is required for recruitment of the DNA repair factor XRCC1, was found to be entirely dispensable. Taken together, these results suggest that DNA repair complex formation and mADPR 2nd messenger accumulation are parallel mechanisms through which activated PARP-1 signals the presence and extent of O/N stress to a cell.


Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Buelow B, Uzunparmak B, Paddock M, Scharenberg AM - PLoS ONE (2009)

PARP-1 synthesizes long, branched chains of poly-mADPR (PAR) on a wide variety of acceptor proteins in the nucleus, notably PARP-1 itself (which appears to accumulate roughly 90% of cellular PAR).Subsequently, PAR is degraded by Poly-ADP-Ribose-Glycosylase (PARG) into mADPR, which is assumed to diffuse out of the nucleus and into the cytosol. There it can bind to the ion channel TRPM2, leading to the influx of cations, including calcium. Additionally, PARP-1 mediates the recruitment of DNA repair proteins to sites of DNA damage through protein-protein interactions (notably through its BRCT domain) and PARylation. The relationship between PARP-mediated mADPR degradation and DNA damage repair remains unclear.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713433&req=5

pone-0006339-g001: PARP-1 synthesizes long, branched chains of poly-mADPR (PAR) on a wide variety of acceptor proteins in the nucleus, notably PARP-1 itself (which appears to accumulate roughly 90% of cellular PAR).Subsequently, PAR is degraded by Poly-ADP-Ribose-Glycosylase (PARG) into mADPR, which is assumed to diffuse out of the nucleus and into the cytosol. There it can bind to the ion channel TRPM2, leading to the influx of cations, including calcium. Additionally, PARP-1 mediates the recruitment of DNA repair proteins to sites of DNA damage through protein-protein interactions (notably through its BRCT domain) and PARylation. The relationship between PARP-mediated mADPR degradation and DNA damage repair remains unclear.
Mentions: Much less is known about the biochemical mechanisms of PARP-1 activation in the context of O/N stress induced formation of mADPR. Compelling evidence indicates that PARP-1-dependent mADPR formation results in mADPR-mediated activation of the TRPM2 Ca2+ channel (Figure 1 and [21]–[24]). However, there are no data addressing the biochemical context in which PARP-1 activation leads to mADPR formation, or the relationship between these mechanisms and PARP-1′s involvement in the DNA damage response. To better define the biochemistry of PARP-dependent mADPR formation, we reconstituted PARP-1 deficient DT40 cells with either WT or various mutant forms of PARP-1 (Figure 2), and determined the capacity of each mutant to support two correlates of O/N stress-induced mADPR formation: NAD degradation and TRPM2 activation. Our results suggest that catalytic activity, DNA binding, and an intact auto-PARylation domain are required for PARP-1-mediated cytosolic mADPR accumulation in vivo. In contrast, the BRCT domain, which is required for recruitment of the DNA repair factor XRCC1, was found to be entirely dispensable. Taken together, these results suggest that DNA repair complex formation and mADPR 2nd messenger accumulation are parallel mechanisms through which activated PARP-1 signals the presence and extent of O/N stress to a cell.

Bottom Line: Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR).To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain.Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

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Related in: MedlinePlus