Radiation Oncology/Radiobiology/DNA Damage Response

DNA Damage Response

Overview

 * Please see the page for more information
 * It is estimated that mammalian cells experience >100,000 DNA lesions per day. The result from replication errors, chemical decay of bases, reactive oxygen species damage, and ionizing radiation
 * Cells have developed DNA repair pathways that are specific for each type of damage, and may depend on which part of cell cycle the damage happens
 * DNA damage is initially recognized by sensor proteins MRN and/or Ku70/80
 * These activate effector molecules, primarily ATM for double strand damage, or in its absence DNA-PKcs. ATR is activated for single strand damage
 * ATM coordinates the DNA damage response (DDR) by several steps
 * Histone H2AX damage foci formation
 * DNA repair machinery activation (NHEJ or HR)
 * Cell cycle checkpoint activation
 * Initiation of apoptosis

Double Strand Damage Sensing
Nijmegen breakage syndrome)]], ATM is unable to initiate damage response
 * DNA damage, particularly in the form of double strand breaks (DSBs) appears to play a key role in radiation-induced cell death. However, it is important to keep in mind that DSBs also occur as part of normal cell process (e.g. DNA replication / fork stalling, programmed rearrangements V(D)J, meiosis), and therefore that mechanisms exists for their repair
 * After radiation exposure, damage-response proteins initiate DNA repair within minutes by localizing to sites of DNA double strand breaks (DSB). The exact sequence has not yet been well established, however, there appear to be 3 pairs of sensors belonging to the PIKK kinase family, which effect >700 proteins
 * MRN -> ATM
 * Ku70-Ku80 -> DNA-PKcs
 * ATRIP -> ATR
 * Several lines of evidence suggest that Mre11-Rad50-Nbs1 complex (MRN) is one of the primary proteins able to sense DSB and begin processing it for repair
 * It appears that short single-strand DNA oligonucleotides are produced as part of the process, via endo- and exonuclease activity
 * Accumulation of these ssDNA oligos appears to constitute a signal of ongoing DSB repair, and induce ATM activity. However, presence of ssDNA oligos alone does not seem sufficient to activate ATM; presence of dsDNA end may be required for ATM activation
 * ATM is detectable at sites of DNA damage within 60 seconds after damage, and persists for up to 4 hours
 * ATM (Ataxia-Telangiectaia-Mutated) is a kinase with multiple downstream actions effected via phosphorylation
 * ATM activation is radiation dose dependent, in response to increasing level of radiation-induced DNA damage. Dose <0.3 Gy induces ATM only weakly, while dose >0.5 Gy produces maximal ATM activation
 * As a first step, ATM autophosphorylates to form the active monomeric form
 * Activated ATM impacts multiple targets (>30) involved in DNA repair, cell cycle control (G1/S, S/G2, G2/M), and apoptosis
 * When NBS1 (part of MRN) is mutated (i.e. in [[Radiation_Oncology/Cancer_Syndromes#Nijmegen_Breakage_Syndrome|
 * Hereditary Syndrome: Ataxia-Telangiectasia (AT)
 * In cells that lack ATM, DNA sensing and phosphorylation of H2AX can occur via the DNA-PKcs pathway
 * This pathway is inactive in cells with ATM present (whether it functions or not)
 * DNA-PKcs becomes activated by Ku70-Ku80 complex, which directly binds to DSB
 * Normal function for DNA-PKcs is in non-homologous end-joining (NHEJ)
 * Finally, ATR plays a role in the repair process as well
 * ATR normally recognizes single-strand breaks and other damage during DNA replication. It may also recognize DNA damage that is unrepaired (alpha component) after DSB
 * However, ATR can become activated after the MRN-ATM complex begins DSB processing
 * Hereditary Syndrome: None because ATR is an essential protein for cell survival

ATM

 * Member of PIKK family (ATM, ATR, DNA-PKcs, mTOR)
 * Exists as an inactive dimer
 * Autophosphorylates on recognition of DNA damage
 * When activated, exists as a monomer
 * It then phosphorylates multiple targets
 * DNA repair: H2AX, NBS1/MRE11 (part of MRN), BRCA1, FANCD2, SMC1 (structural maintenance of chromosome)
 * Cell cycle arrest: p53 (G1 checkpoint), Chk1/2 (S and Early G2 checkpoints), MDC1 (co-localizes with H2AX)
 * Apoptosis: p53, SMAC

H2AX Activation

 * All three effectors (ATM, DNA-PKcs, and ATR) are able to activate histone H2AX to amplify and mark chromatin territory affected by DNA damage
 * This event is regulated by protein MDC1, which spreads H2AX phosphorylation in both directions away from the break. MDC1 also plays a role in activating Chk1 and further S-phase and G2/M phase check points
 * H2AX phosphorylation is thought to alter the chromatin structure, allow access for DNA repair machinery, and act as a docking site for the repair proteins

Cell Cycle Checkpoints

 * There are 4 distinct cell cycle checkpoints in response to DNA damage
 * Mutations in genes responsible for G1, S, and Early G2 checkpoints do not appear to increase radiation sensitivity to single fraction (but may due to delays and reassortment impact multifraction sensitivity)
 * Mutations in genes responsible for Late G2 appear to cause radiosensitization, perhaps by allowing entry into mitosis and resulting in mitotic death
 * There is a dose-rate effect, such that at very low dose rates (<0.01 G/min) there is minimal checkpoint activation, probably due to minimal ATM activation. At ~1 Gy/hr, only Late G2 checkpoint is triggered, leading to reassortment of cells at the G2/M interface (and resulting in the inverse dose rate effect). At higher dose rates, cell cycle progression is inhibited at all checkpoints
 * Please also see the cell cycle page

G1 checkpoint

 * Decision of the cell to initiate cell division
 * In cycling cells, controlled by activation of E2F transcription factor
 * In G0/G1, E2F is bound and inactivated by Rb protein
 * DNA damage activates ATM, which subsequently activates p53 by phosphorylating it and dissociating it from MDM2
 * p53 in turn upregulates p21
 * p21 inhibits cyclinD-CDK4/6 and cyclinE-CDK2
 * These cyclins therefore do not phosphorylate Rb protein, which remains bound to E2F, thus inactivating it
 * When a cell decides to enter into cell cycle, cyclinsD/E phosphorylate Rb, which then dissociates from E2F
 * E2F is a DNA transcription factor, which triggers entry into S phase
 * Does not increase radiation sensitivity
 * Summary: damage - ATM up - p53 up - p21 up - CDK4/6 and CDK2 down - Rb down - E2F down - no entry to S phase

S checkpoint

 * The importance of this checkpoint is to prevent replication forks from replicating through DNA strand breaks
 * CDK2 kinase must remain dephosphorylated for S phase to progress
 * DNA damage activates ATM, which subsequently activates Chk2 kinase (and to lesser degree Chk1)
 * The Chk1/2 kinases then phosphorylate CDC25A, leading to its degradation
 * This increases levels of phosphorylated CDK2 kinase, and slowed progression through S-phase by failure to load CDC45 onto chromatin
 * Lack of CDC 45 prevents recruitment of DNA polymerase α and subsequent replication
 * There also appears to be a pathway from ATM via NBS1 to CDC45, and BRCA1/BRCA2 and FANC-D2 also play some role in this process
 * Does not increase radiation sensitivity
 * Summary: damage - ATM up - Chk2 up - CDC25A down - CDK2 phosphorylated - CDC45 down - slow S phase progression

Early G2 checkpoint

 * Activated by low doses (<1 Gy) of radiation
 * CDK1 kinase must remain dephosphorylated for G2 to continue
 * DNA damage activates ATM and ATR, which activate Chk1 and Chk2 kinases. There is also a pathway from ATR via BRCA1 to Chk1
 * The Chk1/2 kinases then phosphorylate CDC25C, leading to its destruction. The 14-3-3 protein plays a role in this process
 * Destruction of CDC25C leads to increased levels of phosphorylated CyclinB/CDK1 kinase, which blocks cell in G2. CyclinB/CDK1 kinase must be dephosphorylated to enter mitosis
 * Does not increase radiation sensitivity
 * Summary: damage - ATM up - Chk1/Chk2 up - CDC25C down - CDK1 phosphorylated - no mitosis

Late G2 checkpoint

 * Most regulated of all checkpoints
 * Strongly radiation dose-dependent, and effect may last for many hours
 * Independent of ATM, triggered by ATR
 * Different trigger suggests that a different type of DNA damage is checked than double strand break monitored by ATM. It is probably residual DNA damage after repair has been attempted
 * DNA damage activates ATR, which activates Chk1 kinase
 * The Chk1/2 kinases then phosphorylate CDC25C, leading to its destruction. The 14-3-3 protein plays a role in this process
 * Destruction of CDC25C leads to increased levels of phosphorylated CyclinB/CDK1 kinase, which blocks cell in G2. CyclinB/CDK1 kinase must be dephosphorylated to enter mitosis
 * Increases radiation sensitivity, partly due to cells that cannot repair all of their damaged DNA prior to cell division will experience a higher rate of mitotic death

p53

 * p53 is normally degraded via MDM2 ubiqiutin pathway as soon as it is produced
 * In times of cellular stress or DNA damage, p53 becomes activated (phosphorylation and acetylation) and is protected from degradation
 * Cellular stress via MAPK kinases
 * DNA damage via ATM, ATR, CHK1/CHK2, DNA-PKcs as above
 * p53 can also be activated after suppression of MDM2 by p19ARF -> increased p53 levels
 * Activated p53, as the "guardian of the genome", has several roles:
 * Arrests cells at G1 checkpoint via p21 (see above)
 * Potentiates the DNA repair machinery via GADD45 and XP genes
 * Activates the apoptotic pathway via members of the bcl-2 family (Bax, Bak, Bim, Puma) and the intrinsic apoptosis pathway
 * Increases levels of MDM2 for a negative feedback loop
 * The p53 choice between cell cycle arrest + DNA damage repair vs apoptosis is not well understood

DNA Repair

 * Please see the DNA repair page