Genome quality control and repair is controlled by multiple signaling pathways and repair mechanisms. Sensing DNA damage results in the initiation of a number of programs, including but not limited to cell cycle arrest, checkpoint activation, DNA damage repair, transcription of effectors, autophagy, chromatin remodeling and, as a last resort, apoptosis (3). Defects in the DNA damage response (DDR) pathways are associated with a number of disorders that predispose individuals to cancer as well as other defects. For example, individuals with Ataxia telangiectasia (A-T) have a mutation in ATM, a major signal transducer of the DDR, which is associated with defects in double stand break (DSB) repair. Patients with A-T are sensitive to ionizing radiation, have immune defects, develop neurodegeneration and are predisposed to developing lymphomas (4).
The source of the DNA damage and the type of lesion produced determines the kind of DDR that will be dispatched. The DDR is initiated by binding of sensors to lesions within the DNA. DDR components are recruited to the sites if damage, forming foci. The DNA damage is thought to be sensed by alterations in the chromatin structure arising from the lesion. In the case of DSBs, the ATM-ATR-DNAPK pathway induces the phosphorylation of the histone variant γH2AX on the chromatin flanking the DSB sites. This results in ubiquitin mediated recruitment of DSB repair factors and additional chromatin modifying factors which act to amplify the DNA damage signal, recruiting even more repair associated proteins (3–5).
There are five main DDR mechanisms: mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), nonhomologous end joining (NHEJ), and homologous recombination (HR). MMR, BER, and NER are used for different types of base associated lesions that require a single strand incision. NEHJ and HR repair mechanisms are involved with DSB repair (3–5) (see figure DNA damage repair pathways
With MMR, the mismatch creates a loop in the DNA which is recognized and repaired by nuclease mediated single strand incision followed by polymerase and ligase activity. In BER, the damaged base is recognized and removed by DNA glycosylase. The lesion is repaired by nuclease, polymerase and ligase activity. NER is initiated by recognition of lesions that distort the helical structure of the DNA. There are two kinds of NER: global genome NER which surveys the whole genome for helix distortion and transcription coupled NER which targets lesions that block transcript elongation. A key difference between NER and the other single strand DNA (ssDNA) repair systems is the excision of a 22-30 base oligo, rather than removal of a single base. The remaining ssDNA is then repaired by the DNA replication machinery (3–5).
DSBs occur from exposure to ionizing radiation, free radicals, chemicals, and from replication of a single strand break. DSBs are repaired via HR or NHEJ. HR uses the sister chromatid as the template for repair and therefore can only occur during S phase or G2. HR is initiated by the MRE11-RAD50-NBS1 complex which binds to and processes the ends of the damaged DNA, generating ssDNA. The ssDNA invades the template and repair is mediated by polymerase, nuclease, helicase, and ligase activity, followed by resolution of the structure (3–5).
NHEJ can occur at any point in the cell cycle. This mechanism of repair is initiated by recognition and processing of the DSB by the Ku70/Ku80 heterodimer. This activates DNA-PKcs which mediates recruitment of end processing enzymes and polymerase. DNA ligase IV mediates re-ligation of the broken ends of the DNA. This method of repair is error prone and results in sequence deletion. PARP1/2 can also sense DSBs, promoting an alternative NHEJ repair pathway (3–5).
The DDR is critical for maintaining the integrity of the genome, but like all good things, moderation is the key. The DDR must be activated in response to damage, but then must be turned off to prevent dysregulated activity. The activity of different cellular processes is often controlled by post-translational modifications. Ubiquitination, sumoylation, phosphorylation, and acetylation are examples of post translational modifications that expand the function of the proteome by changing the activity of the modified protein (4). A common way to "shut off" a process is to induce the degradation of a key component of the pathway. Robert et al provides evidence of acetylation regulated degradation of Sae2, a key mediator of DSB repair, via autophagy (2).
Autophagy was initially identified as a mechanism for recycling energy through self digestion of cytoplasmic contents in times of stress and/or starvation. This is now a well recognized mechanism for turnover of long lived proteins, protein aggregates and damaged organelles. Through a series of enzymatic reactions regulated by the autophagy genes (ATGs), cytoplasmic cargo is delivered to the lysozome inside a double membrane vesicle.
There are four main steps to autophagy: 1. vesicle nucleation; formation of an isolation membrane 2. vesicle elongation and completion; growth and closure, 3. fusion of the autophagosome with the lysosome, forming an autolysosome, and 4. degradation of the vesicle and its contents. These steps are executed by the ATG proteins which respond to signals from the mTOR kinase. mTOR kinase shuts off autophagy in the presence of growth factors and abundant nutrients. Inhibition of mTOR results in induction of autophagy (6).
Robert et al inhibited histone deacetylase (HDAC) activity, resulting in hyperacetylation of proteins. After exposure to DNA damage agents they observed an absence of DDR. This correlated with failure to repair DSBs. A number of proteins associated with DSB repair are known to be acetylated, but the function of this post translational modification was not known. Robert et al noticed decreased association of Sae2 and Exo1 with DSB ends and decreased levels of Sae2 protein. Inhibition of mTOR activity with rapamycin, thus triggering autophagy also resulted in decreased levels of Sae2 (2).
This evidence presented by Roberts et al suggests that this may be a mechanism for keeping DNA repair enzymes away from cellular DNA that isn't damaged, preventing accidental "repair" of replicating DNA. Severely damaged DNA has been shown to accumulate at nuclear pores. Perhaps the severely damaged DNA and associated machinery are removed from the nucleus via an autophagic process regulated by the acetylation status of key repair proteins (see figure Regulation of DNA repair by autophagy
The cell has evolved a number of mechanisms for protecting our DNA from the constant onslaught of potential mutagens. It is important, however to regulate the activity of these DNA repair enzymes. The report published by Robert et al suggests a novel mechanism for regulating the activity DSB repair enzymes through an acetylation dependent autophagic process. This observation opens up the world of autophagy and HDAC inhibitors as targets for anti-cancer drug development. Many questions still remain. Multiple DDR proteins are acetylated. Is this mechanism of regulation limited to Sae2 or is this a general mechanism for DDR proteins? How does acetylation signal autophagy? Could "shutting off" the DDR sensitize cancer cells to existing anti-cancer DNA damage inducing drugs? Does DNA damage result in activation of pathways regulating autophagy and HAT and HDAC activity? Is sensitivity to DNA damage inducing cancer drugs influenced by polymorphisms in autophagy related genes? There is a link between decreased autophagy and cancer. Is the autophagic pathway dysregulated in cancer, resulting in inappropriate DNA repair and cell transformation?
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