Biology Assays & Protocols
- Transfection protocol
- Flow cytometry assay
- Apoptosis assay
- PI3K-Akt signaling pathway
Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes including primarily DNA repair and programmed cell death (apoptosis).
The PARP family consists of 17 members. They have all very different structures and functions in the cell.
- PARP1, PARP2, VPARP (PARP4), Tankyrase-1 and -2 (PARP-5a or TNKS, and PARP-5b or TNKS2) have a confirmed PARP activity.
- Others include PARP3, PARP6, TIPARP (or "PARP7"), PARP8, PARP9, PARP10, PARP11, PARP12, PARP14, PARP15, and PARP16.
A typical PARP molecule is composed of four domains: a DNA-binding domain, a caspase-cleaved domain(see below), an auto-modification domain, and a catalytic domain. The DNA-binding domain is composed of two zinc finger motifs. In the presence of damaged DNA (base pair-excised), the DNA-binding domain will bind the DNA and induce a conformational shift independent of the other domains. The auto-modification domain is responsible for releasing the protein from the DNA after catalysis. Also, it plays an integral role in cleavage-induced inactivation.
PARP is found in the cellular nucleus. Its major role is to detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair. PARP activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After repairing, the PAR chains are degraded via PAR glycohydrolase (PARG).
It is interesting to note that NAD+ is required as substrate for generating ADP-ribose monomers. The overactivation of PARP may deplete the stores of cellular NAD+ and induce a progressive ATP depletion, since glucose oxidation is inhibited, and necrotic cell death. In this regard, PARP is inactivated by caspase-3 cleavage (in a specific domain of the enzyme) during programmed cell death.
PARP enzymes are essential in a number of cellular functions, including expression of inflammatory genes: PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF.
Role in cell death
Upon DNA cleavage by enzymes involved in cell death (such as caspases), PARP can deplete the ATP of a cell in an attempt to repair the damaged DNA. ATP depletion in a cell leads to lysis and cell death. PARP also has the ability to directly induce apoptosis, via the production of PAR, which stimulates mitochondria to release AIF. This mechanism appears to be caspase-independent.
Role in repairing DNA nicks
One important function of PARP is assisting in the repair of single-strand DNA nicks. It binds sites with single-strand breaks through its N-terminal zinc fingers and will recruit XRCC1, DNA ligase III, DNA polymerase beta, and a kinase to the nick. This is called base excision repair (BER). PARP-2 has been shown to oligomerize with PARP-1 and, therefore, is also implicated in BER. The oligomerization has also been shown to stimulate PARP catalytic activity. PARP-1 is also known for its role in transcription through remodeling of chromatin by PARylating histones and relaxing chromatin structure, thus allowing transcription complex to access genes.
Role of tankyrases
The tankyrases are PARPs that comprise ankyrin repeats, oligomerization domain (SAM), and a PARP catalytic domain (PCD). Tankyrases are also known as PARP-5a and PARP-5b. They were named for their interaction with the telomere-associated TRF1 proteins and ankyrin repeats. They may allow the removal of telomerase-inhibiting complexes from chromosome ends to allow for telomere maintenance. Through their SAM domain and ANKs, they can oligomerize and interact with many other proteins, such as TRF1, TAB182 (TNKS1BP1), GRB14, IRAP, NuMa, EBNA-1, and Mcl-1. They have multiple roles in the cell, vesicular trafficking through its interaction in GLUT4 vesicle (GSVs) with insulin-responsive amino peptidase (IRAP). It also plays a role in spindle assembly through its interaction with nuclear mitotic apparatus (NuMa), therefore allowing bipolarity. In t
he absence of TNKs, mitosis arrest is observed in pre-anaphase through Mad2 kinetochore checkpoint. TNKs can also PARsylate Mcl-1L and Mcl-1S and inhibit both their pro- and anti-apoptotic function. Relevance of this is not yet known.
Role in Epigenetic DNA modification
PARP-mediated post-translational modification of proteins such as CTCF can affect the amount of DNA methylation at CpG dinucleotides. This regulates the insulator features of CTCF can differentially mark the copy of DNA inherited from either the maternal or the paternal DNA through the process known as genomic imprinting. PARP has also been proposed to affect the amount of DNA methylation by directly binding to the DNA methyltransferase DNMT-1 after attaching poly ADP-ribose chains to itself after interaction with CTCF and affecting DNMT1's enzymatic activity .
Apoptotic PARP cleavage assay:
1. Western-blot analysis of PARP1
- Harvest cultured cells with lysis buffer.
- Quantify total protein concentration by BCA assay.
- Isolate target protein by SDS-PAGE.
- Transfer proteins to nitrocellulose or PVDF membranes.
- Incubate in blocking buffer (PBST or TBST with 1% bovine serum albumin or 1% non fat milk), for 20 minutes to 1 hr at RT or 37°C.
- Incubate with 1:1000~1:2000 of PARP1 monoclonalantibody (MAb) or PARP1 polyclonal antibody (PAb) at RT for 1~2h or at 4°C for overnight.
- Wash the membrane with PBST or TBST for 3*10min
- Incubate with 1:5000~1:10000 of anti-human IgG conjugated with HRP or AP at RT for 30min~1h
- Wash the membrane with PBST or TBST for 4*15min
- Develop with ECL or NBT/BCIP