Studying DNA Damage And Repair

Extensive research on DNA damage has revealed an intricate network of DNA damage sensing and signaling pathways. The molecular networks that comprise the cell's DNA damage responses are complex and involve proteins acting as sensors, transducers and effectors for the different pathways. Different proteins are required to transduce the damage signals and execute the response - cell cycle arrest, DNA repair and apoptosis. The Nbs1/Mre11/Rad50 (MRN) complex is recruited to DNA double strand breaks (DSBs) forming DNA damage foci together with BRCA1, MDC1 and 53BP1. The transducer for DSBs is the kinase ATM (Ataxia-Telangiectasia Mutated), which switches on a variety of effector proteins, including p53, MDM2 and CHK2. In contrast, stalled replication forks and single strand breaks (SSBs) trigger ATR (Ataxia-Telangiectasia and Rad3 related) activation, which switches on p53 and CHK1. These effector proteins then regulate cell cycle progression and arrest, apoptosis and cell senescence.

Cell cycle Checkpoint Regulation

Cell cycle checkpoints function to ensure that the cell's DNA is intact and that the critical stages of the cell cycle are completed before continuing on to the next stage. In response to DNA damage, ATM/ATR kinases activate the checkpoint serine/threonine kinases CHK1 and CHK2, which target cdc25A leading to its ubiquitin-mediated proteolysis and cell cycle arrest in the G1/S transition. Also, CHK1 and CHK2 activation phosphorylates all cdc25 phosphatases sequestering it away from the cdk2-cyclinA and cdk1-cyclinB, which regulate progression into S phase and the G2/M transition, respectively. Additional regulators of the G2/M transition include the Polo-like kinases (PLK) and Aurora-like kinases. In the presence of extensive DNA damage, p53 activate genes to trigger apoptosis. The dynamic spatio-temporal regulation of the DNA damage response network remains to be elucidated.

DNA Repair Pathways

DNA plays a critical role as a repository of hereditary information. However, many environmental factors and endogenous cellular processes result in a high frequency of DNA damage. DNA repair mechanisms are essential for genomic stability, maintenance of proper cellular function and survival for all organisms. Eukaryotic cells have developed various pathways for DNA repair. In humans, DNA damage is involved not only in tumor formation and aging but also a variety of genetically-inherited disorders including Xeroderma pigmentosum (XP), Cockayne's syndrome (CS), trichothiodystrophy (TTD), and hereditary non-polyposis colon cancer (HNPCC).

DNA repair mechanisms to fix the different types of DNA damage are essential for genomic stability, maintenance of proper cellular function and survival for all organisms. Eukaryotic cells have developed various pathways for DNA repair. Different DNA repair mechanisms are available for the cell to combat the various types of lesions. Some injuries are corrected by direct reversal while most DNA repair events are mediated by different proteins. The different repair pathways include single-strand break repair (SSBR), mismatch repair (MMR), base-excision repair (BER), nucleotide excision repair (NER) and double strand break repair (DSBR). In DSB, two repair mechanisms are involved, non-homologous end-joining (NHEJ) and homologous recombination (HR). Currently, new proteins are being identified as part of the cell's response to DNA damage. A recent article in Science (Cotta-Ramusino et al. 2011) reports a novel protein, RHINO (MGC13204), recruited to sites of DNA damage and it is involved in ATR and checkpoint activation. Another report from Ozeri-Galai et al. (Mol Cell 2011) shows that the basis for DNA fragility is replication fork stalling at AT-rich sequences and the inability to activate additional origins under replication stress.

Antibodies to study DNA damage

DNA damage antibodies are used in localizing nuclear foci or DNA damage heterochromatin foci. ATM antibody and antibodies against the different proteins involved in the DNA damage response (or DNA damage antibodies) are readily available and used by researchers in the lab. DNA damage antibody sampler kits are also available, usually packaged with primary antibodies and the corresponding secondary antibodies, giving a better value for researchers studying the pathways involved in the cellular DNA damage response. Popular antibodies include the ATM antibody, CHK1/CHK2 phospho-specific antibodies and the ICC/IF validated DNA damage antibodies.

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