Authors: Rehna Krishnan, Deepan Narayanan, Afzal Husain
Why should we study DNA Damage Repair?
DNA is the carrier of genetic information and is constantly exposed to insults leading to DNA lesions. These lesions can be from endogenous sources like replication stress, hydrolysis, oxidation and alkylation or exogenous sources such as ionizing or ultraviolet radiation and various chemical agents present in the environment. Lesions cause structural damage to DNA molecules, which can dramatically change the expression and functions of encoded genes. Replication of damaged DNA propagate mutant protein and can ultimately lead to disease. For example, frequent TP53 somatic mutations in many cancer types can disrupt the DNA damage response, apoptosis, or senescence pathways active in many early-stage cancers. Thus, DNA repair mechanisms need to be intricately regulated in order to detect and fix damage and maintain genomic stability. Thus, loss of DNA repair function is an important determinant of cancer risk, progression, and therapeutic response. Availability of high quality ChIP-validated antibodies is critical to understand various roles of DNA repair proteins in the maintenance of genomic instability and tumorigenesis.
What are the various pathways involved in Double Strand Break (DSB) repair?
Double strand breaks are extremely harmful to cells because they can lead to cell death if not repaired. DSBs can cause deletions, translocations and fusions in DNA, and are hallmark of cancerous cells.
DSBs are mainly repaired by two main pathways:
- Homologous recombination (HR) – when DSBs arise during S-phase or after replication, a second DSB repair pathway, the HR pathway, can come into action. In contrast to NHEJ, HR is error free, and requires not only homologous DNA sequences as repair templates, but also ssDNA ends to search for homologous sequences (2). HR pathway plays an important role in repairing the DSBs produced by replication fork collapse (3).
- Non-homologous end joining (NHEJ) – when DSBs arise in mammalian cells before DNA replication, the NHEJ pathway, a first line of defense, gets activated. Although it tends to be error prone, NHEJ is a quick and robust way to rejoin DNA ends throughout the cell cycle.
DNA repair is regulated by a series of enzymes including kinases, ligases and polymerases (4-5). Understanding the molecular mechanism behind DNA damage repair pathway and its regulation is crucial when studying the effects of a dysfunctional DNA damage response pathway. To help accelerate DNA repair research, Invitrogen offers specific antibodies against some of these key targets.
How is Double Strand Break repair regulated by ATM kinases?
ATM and ATR, the members of phosphatidylinositol 3-kinase-like protein kinase (PIKKs) family, act as critical regulators of the damage response to double strand breaks and replication stress. ATM is primarily activated by DNA damaging agents and signals both the DNA repair machinery and cell cycle checkpoints (5). Once activated, ATM phosphorylates mediator proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Chromatin Immunoprecipitation (ChIP) is used to asses ATM activation as well as recruitment of various mediator of DNA damage repair at the site of DSB (Figure 1A).
What are the major markers of the DNA damage?
When cells are exposed to endogenous or exogenous DNA damaging agents, it forms double-strand breaks, which is followed by the phosphorylation of a variant of histone H2A, H2AX, by ATM and ATR kinases (6). The phosphorylated H2AX (γH2AX; gamma-H2AX) helps in the recruitment of various DNA repair proteins to the DNA damage sites, and forms gamma-H2AX foci that can be visualized by immunofluorescence (Figure 2) or quantified by ChIP (Figure 1B).
What are the various substrates of ATM involved in HR repair?
- CtIP– DNA end resection is a critical regulatory step in HR repair which commits a cell to repair DNA. During end resection, which is controlled by ATM through CtIP, DSBs are preferentially degraded by nucleases to generates long 3′-ssDNA overhangs. CtIP associates with BRCA1 in S and G2 phases which ubiquitinates it and facilitates its association with damage sites (7). Recruitment of CtIP, as assessed by ChIP, can be used to assay the contribution of HR pathway in DNA damage repair (Figure 1C).
- BRCA1/2 -The function of BRCA1/2 in HR repair is to regulate the binding of RAD51 which in turn catalyzes homologous pairing and DNA strand exchange at the break site (8). BRCA1/2 promotes the removal of 53BP1, a target of ATM kinase, in S phase to allow DNA end resection. The BRCA1/2 dependent removal of 53BP1 facilitate the transition from NHEJ to HR. BRCA1/2 ChIP assay can be used to ascertain that the DNA damage is repaired by the HR pathway.
What are the various substrates of ATM involved in NHEJ repair?
- KU70/Ku80, which rapidly binds to DSBs and possesses DNA end processing activity, acts as an independent sensor of DSBs. Once recruited to the double strand break site, Ku activates DNA-Pkcs to initiate NHEJ (9). DNA-PKcs, an enzyme encoded by PRKDC or XRCC7 gene, is a serine/threonine protein kinase. Ku or DNA-Pkcs antibodies are used as a marker for NHEJ DNA break repair.
- ERCC1-XPF is a structure specific endonuclease involved in nucletide excision repair and in double strand break repairs. In addition to it its critical role in the removal of interstrand cross-links (ICL) induced upon UV exposure, it is also important for NHEJ (10). Its binding to the 3’-overhangs can be assessed by ChIP as seen in Figure 1D.
Learn more about ChIP Validation
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Drivers of the Chromosomal Passenger Complex
Figure 1 Chromatin Immunoprecipitation (ChIP) was performed using A) Anti-Phospho-ATM (Ser1981) antibody (Product # MA5-15185) using primers binding to active (GAPDH and ACTB) and inactive (SAT2 satellite repeats and SAT alpha) loci. D) Anti-H2AX antibody (Product # PA1-41004) using primers binding to active (GAPDH) and inactive (MYOD, SAT2 and SATA satellite repeats) loci. C) Anti-CtIP antibody (Product # PA5-21983) using primers binding to active (GAPDH and CDKN1A) and inactive (SAT2 satellite repeats) loci. D) Anti-XPF antibody (Product # MA5-12060) using primers binding to active (EE1F1A and GAPDH) and inactive (SAT alpha) loci.
Figure 2: Immunofluorescence analysis of HeLa cells using Phospho-Histone H2AX-S139 Polyclonal antibody (Product # PA5-97354). Blue: DAPI for nuclear staining. HeLa cells were treated by UV for 15-30 minutes at RT (left). Blue: DAPI for nuclear staining.
Table 1: ChIP-validated antibodies for DNA repair proteins research
| SKU | Target Name | Repair Pathway Involved | Applications Tested |
| MA1-7629 | p53 Monoclonal Antibody | HR and NHEJ Repair | IF, ICC |
| PA5-27822 | p53 Polyclonal Antibody | HR and NHEJ Repair | WB, IF, ICC |
| MA5-15031 | PARP Monoclonal Antibody | Base excision repair | WB, IF, ICC |
| PA5-27219 | PARP Polyclonal Antibody | Base excision repair | WB, IF, ICC |
| PA5-34803 | PARP Polyclonal Antibody | Base excision repair | WB, IF, ICC |
| PA5-29278 | RUVBL1 Polyclonal Antibody | HR Repair | WB, IF, ICC |
| PA5-76147 | FANCI Polyclonal Antibody | HR Repair | WB, IF, ICC |
| PA5-59014 | FANCI Polyclonal Antibody | HR Repair | WB, IF, ICC, IHC |
| MA5-13110 | Ku70 Monoclonal Antibody | NHEJ Repair | WB, IF, ICC, IHC |
| MA5-15185 | Phospho-ATM (Ser1981) Monoclonal Antibody | HR and NHEJ Repair | WB, IF, ICC |
| PA5-21983 | CtIP Polyclonal Antibody | HR Repair | WB |
| MA1-23164 | BRCA1 Monoclonal Antibody | HR Repair | WB, IF, ICC, IHC |
| PA5-18498 | Ku80 Polyclonal Antibody | NHEJ Repair | WB, IF, ICC, IHC |
| MA5-12060 | XPF Monoclonal Antibody | NHEJ Repair | WB |
| 701953 | Histone H2A.X Recombinant Rabbit Monoclonal Antibody | HR and NHEJ Repair | WB, IF, ICC |
| PA1-41004 | Histone H2A.X Polyclonal Antibody | HR and NHEJ Repair | WB, IHC |
| PA5-97354 | Phospho-Histone H2A.X (Ser139) Polyclonal Antibody | HR and NHEJ Repair | WB, IF, ICC, IHC |
References
1) Mazouzi, A, Battistini, F, Moser, S.C, Ferreira da Silva, J, Wiedner, M, Owusu, M et al., (2017) Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH. Mol Cell. 68(4), 797-807
2) Maréchal, A., & Zou, L. (2013). DNA damage sensing by the ATM and ATR kinases. Cold Spring Harbor perspectives in biology, 5(9), a012716.
3) Meena Shrivastav, Leyma P De Haro & Jac A Nickoloff. (200) Regulation of DNA double-strand break repair pathway choice. Cell Research. 18, pages 134–147.
4) Jiricny, J. (2006). The multifaceted mismatch-repair system. Nat. Rev. Mol. Cell Biol. 7, 335–346.
5) Lindahl, T., and Barnes, D.E. (2000). Repair of endogenous DNA damage. Cold Spring Harb. Symp. Quant. Biol. 65, 127–133.
6) Kinner, A., Wu, W., Staudt, C., & Iliakis, G. (2008). Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic acids research, 36(17), 5678–5694.
7) Zhou, B.B., and Elledge, S.J. (2000). The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439.
8) Holloman W. K. (2011). Unraveling the mechanism of BRCA2 in homologous recombination. Nature structural & molecular biology, 18(7), 748–754.
9) Mahaney, B.L., Meek, K., and Lees-Miller, S.P. (2009). Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem. J. 417, 639–650.
10) J. 417, 639–650.Ahmad, A., Robinson, A.R., Duensing, A., van Drunen, E., Beverloo, H.B., Weisberg, D.B., Hasty, P., Hoeijmakers, J.H., Niedernhofer, L.J. (2008) ERCC1-XPF endonuclease facilitates DNA double-strand break repair. Mol. Cell. Biol. 28 :5082–5092.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
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