During the DNA damage response (DDR), a cascade of signaling events occurs that include DNA damage detection, cell cycle arrest to facilitate repair, and activation of DNA repair pathways (homologous recombination, non-homologous end joining, etc.). The DNA lesion is either repaired or cell apoptosis occurs if the damage is irreparable.
Certain DNA damage response proteins are responsible for detection of DNA damage and facilitation of downstream DNA repair mechanisms. Ataxia-Telangiectasia Mutated (ATM) activates in response to DNA double-stranded breaks (DSBs) and phosphorylates downstream targets to initiate DNA damage repair. DNA-dependent protein kinase (DNA-PK) is also involved in recognition of DNA DSBs and initiates repair mechanisms. ATM and Rad3-related (ATR) activates in response to single-stranded breaks and stalled replication forks, playing an important role in the response to DNA replication stress.
Various sensor, signaling, and effector proteins attend to different kinds of DNA damage. Single-strand breaks (SSBs) in DNA are mended by either base excision repair (BER), nucleotide excision repair (NER), or mismatch repair (MMR). Double stranded breaks (DSBs), the most serious type of DNA damage, are mostly remedied by homologous recombination (HR) or non-homologous end-joining (NHEF).
DNA-PK (DNA-Dependent Protein Kinase)
Double-stranded breaks (DSBs) are a severe form of DNA damage that occurs from various sources, such as ionizing radiation or chemicals. Upon recognition of a DSB, DNA-PK binds to the broken ends and orchestrates downstream DNA damage repair mechanisms through non-homologous end joining (NHEJ). The recognition of DSBs by DNA-PK has also been shown to activate Type I interferon responses. DNA-PK acts similarly to a pattern-recognition receptor (PRR) by recognizing DSBs and then triggering or altering inflammatory responses.
PARP1 (Poly (ADP-ribose) Polymerase 1)
PARP1 is the principal first responder to DNA strand breaks. It interacts with chromatin structure and various DNA repair effectors to select both the appropriate repair pathway for the damage involved and ensures efficiency of the repair. PARP1 performs Poly(ADP-ribosyl)ation or PARylation by synthesizing long and branched negatively charged polymers of ADPR onto acceptor proteins. In excess, PARP1 expression can lead to genomic fragmentation and cell death. It is upregulated in several cancer types, including ovarian, hepatocellular, colorectal, and leukemia.
PARG (Poly (ADP-ribose) Glycohydrolase)
PARG is responsible for “resetting” PARP1 and DNA repair effectors after DNA repair has been completed. As such, PARG functions to regulate the repair of SSBs, DSBs, and replication forks. Upon completion of the repair, PARG hydrolyzes pADPR on PARP1 and similarly tagged associated proteins to diminish intracellular pADPR and dissemble the repair machinery at a given site. This reactivates the previous heavily poly ADP-ribosylated PARP1. Without PARG, proper PARP1 activity is severely curtailed, leading to accumulating DNA damage. While PARG upregulation impedes tumor growth in a canonical manner, PARG inhibitors can cause replicative fork failure and cancer cell death.
Measure PARG Activity with the PARG Assay
Robust PARG Assay (Z’ > 0.8) Measures Enzyme Activity
POLQ (DNA Polymerase Theta)
POLQ regulates the microhomology-mediated end-joining (MMEJ) pathway of DSB repair. POLQ joins 5′-resected DNA substrates by locating and pairing microhomologies present in 3′-overhanging single-stranded tails, cleaving the extraneous 3′-DNA by dNTP-dependent end-processing, and then extending the nascent 3′ end from the microhomology annealing site. In effect, POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks. In prostate and and pancreatic cancer, the canonical HR repair mechanisms are impaired, leading to overexpression of and almost complete dependence upon POLQ.
Discover POLQ Inhibitors with the POLQ Helicase Assay
WRN (Werner Syndrome Helicase)
WRN is involved in several processes that ensure genomic integrity: DNA replication, base excision repair, and the DDR. WRN is the only human RecQ helicase to possess an exonuclease capability. This added function allows WRN to stabilize replication forks and effect DSB repair via HR, NHEJ, and MMEJ. Deficient or impaired production of WRN, whether through nonsense/missense mutation or promoter methylation, produces accelerated senescence and a greatly increased risk of colorectal and gastric cancer. Nonetheless, cancers that lack adequate mismatch repair machinery and possess enhanced mutation at microsatellite repeats (MSI-H) depend upon upregulated WRN for survival.
Discover WRN Helicase Inhibitors with the WRN Helicase Assay
Discover WRN Exonuclease Inhibitors with the WRN Exonuclease Assay
There is extensive crosstalk between the DDR pathways and innate immune pathways. In this illustration, we highlight these interconnections between the pathways. Here, DNA damaging agents induce a DDR in the tumor cell’s nucleus. dsDNA forms in the cytoplasm from leaked DNA from the nucleus or micronuclei. Detection of the dsDNA by PRRs is accompanied by an innate immune response, inducing Type I IFNs and pro-inflammatory cytokines. Extracellularly, adenosine (ADO) and cGAMP regulate innate immune responses; the levels of both are controlled by ectonucleotidases.
In our latest eBook, we detail connections between DDR & innate immunity pathways, and describe Transcreener Assays and Assay Systems for key therapeutic targets.
Synthetic lethality is when the occurrence of two genes together is lethal, but the occurrence of those genes individually is not lethal. Using the synthetic lethal strategy for anti-cancer drug therapeutics has shown tremendous promise: tumors with genetic deficiency in the DDR are selectively killed with a drug targeting a second DDR protein. The use of PARP inhibitors for ovarian tumors with BRCA-1 mutations was the first clinical example of this approach. More recently, POLQ was found to be synthetically lethal in tumors with common BRCA and ATM mutations, providing an alternative therapeutic strategy for PARPi-resistant tumors.
DNA damage response assays play a crucial role in investigating the DNA damage response proteins, and developing small molecule modulators for the pathway that show great potential as disease therapeutics. Here at BellBrook, we develop and commercialize ready-made assays for DDR proteins that will accelerate researchers’ efforts to discover these modulators.
We focus on creating complete assay solutions for tougher enzyme targets that are difficult to source enzyme or develop an assay for so you can focus on optimizing your lead molecules. We also offer lead discovery and assay development services for those who do not want to bring an assay in-house. With our services, you will directly collaborate with our scientists who are experts in the DDR pathway. Our services offer quick turnaround times, custom assay conditions, and extensive expertise.
A simple assay protocol that is ready to scale up for HTS (Z’ > 0.7).
Many of our assays offer direct nucleotide detection, which reduces the risk of false positives or missing a hit.
The unparalleled sensitivity of the assay technology allows high-throughput screening with reduced enzyme usage.
Stability at room temperature for at least 8 hours ensures data quality doesn’t suffer in a large, automated screen.
Find the right resources to help you get to your solution faster.
Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.
Gupta, P, et al. (2022) Enigmatic role of WRN-RECQL helicase in DNA repair and its implications in cancer. Journal of Translational Genetics and Genomics, 6(2): 147-56. http://dx.doi.org/10.20517/jtgg.2021.60
James, D. I. et al. (2016) First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib. ACS Chemical Biology, 11 (11), 3179-3190. https://doi.org/10.1021/acschembio.6b00609
Molinaro, C. et al. (2021) Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies. Cancers, 13(15), 3819. https://doi.org/10.3390/cancers13153819
Mullard, A. (2022) What’s next for the synthetic lethality drug discovery engine? Nature Reviews Drug Discovery, 1474-1784 (online). https://doi.org/10.1038/d41573-022-00107-0
Nijman, S. (2011) Synthetic lethality: General principles, utility and detection using genetic screens in human cells. FEBS Lett., 585(1):1-6. https://doi.org/10.1016/j.febslet.2010.11.024
Pascal, J.M. (2019) The comings and goings of PARP-1 in response to DNA damage. DNA Repair, 71:177-182. https://doi.org/10.1016/j.dnarep.2018.08.022
Taffoni, C. et al. (2021) Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes. Frontiers in Immunology, 12, 660560. https://doi.org/10.3389/fimmu.2021.660560
Zahn, K.E. and Jensen, R.B. (2021) Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair. Genes, 12(9):1310. https://doi.org/10.3390/genes12091310
Zatreanu, D. et al. (2021) POLQ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance. Nature Communications, 12:3636. https://doi.org/10.1038/s41467-021-23463-8
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
