Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.
The eukaryotic genome is under constant mutational stress through exposure to exogenous and endogenous agents that damage DNA. These external and internal factors damage DNA introducing a wide variety of genetic alterations including: deletions, translocations, and chromosome loss, which can result in cell death. One type of genetic alteration, DNA double-stranded breaks (DSBs), poses a serious threat to cell viability and genome stability if left unrepaired or repaired incorrectly. In order to repair these DSBs, cells have evolved the complex repair pathways: nonhomologous end-joining (NHEJ) and homologous recombination (HR) [
Regulating repair proteins through posttranslational modification, such as phosphorylation, provides molecular switches for regulating DSB repair. Phosphorylation is the addition of a phosphate (PO4) group to a protein in order to induce a conformational change that initiates protein activation or deactivation. In DNA repair proteins, phosphorylation has been found to occur on serine and threonine residues, but it can also occur on tyrosine, histidine, arginine, or lysine residues as well. Phosphorylation of DNA repair proteins generally results in activation of the proteins to facilitate DNA repair. The mechanism of reversible phosphorylation in proteins is an important regulatory mechanism for DNA repair pathways.
The primary goal of nonhomologous end-joining (NHEJ) is to resolve DSBs generated by exogenous and endogenous agents that damage DNA. Key factors for facilitating proper repair through NHEJ include the Ku70/80 heterodimer, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4), DNA ligase IV, and XRCC4-like factor (XLF). As shown in Figure
NHEJ model. After a DSB has occurred due to an exogenous or endogenous DNA damaging agent, the NHEJ repair process commences. In step 1, the Ku70/80 heterodimer recognizes and binds the DSB, which induces inward translocation of Ku and recruits DNA-PKcs to the ends of the DSB to form DNA-PK. In step 2, depending on the type and complexity of the DSB break, the DNA ends are processed by different processing factors such as, Artemis, PNKP, DNA polymerases, or the MRN complex (MRE11/RAD50/NBSI). Also, DNA-PKcs autophosphorylate in this second step and will then dissociate from the DSBs. In the final step, XLF stimulates the XRCC4/DNA ligase IV complex to ligate the DNA ends to repair the DSB.
Researchers have recently implicated DNA-PK as a central regulator of DNA end access via phosphorylation [
The catalytic subunit of DNA-PK, DNA-PKcs, is responsible for the kinase activity. DNA-PKcs is a member of the phosphatidylinositol-3-kinase-like (PIKK) family of serine/threonine protein kinases, which also includes Ataxia Telangiectasia-Mutated (ATM) and ATM- and Rad3-related (ATR) [
Different DNA-PKcs phosphorylation sites have distinct functional consequences. Several studies have discovered two functionally relevant autophosphorylation clusters: ABCDE (T2609, S2612, T2620, S2624, T2638, and T2647) and PQR (S2023, S2029, S2041, S2051, S2053, and S2056) [
Additionally, DNA-PKcs contains a phosphorylation site within the activation loop (T site) of the kinase (T3950). Phosphorylation of the T site influences the DNA ends joining efficiency [
Several studies have associated full kinase activation with the combined DNA-PK complexes rather than a single DNA-PK complex, suggesting that the DNA-PKcs must phosphorylate only in
HR repairs DSBs by using homologous sequences elsewhere in the genome to prime repair synthesis, especially DSBs produced by replication fork collapse [
Phosphorylation events in the initiation of HR. After DNA damage is inflicted, the MRN complex processes the ends of the DSBs. BRCA1 is phosphorylated by ATM and CHK2 and regulates the MRN complex. RPA then associates with the 3′ ssDNA overhangs and becomes phosphorylated. Rad52 binds RPA and displaces it to allow for Rad51 binding. BRCA2 binds to Rad51 until BRCA2 becomes phosphorylated, releasing Rad51 and allowing it to localize to the DSB with Rad52. HR-mediated repair continues: Rad51 then forms a nucleoprotein filament that invades a homologous sequence and activates strand exchange to generate a crossover between the juxtaposed DNA.
Mammalian replication protein A (RPA) is a DNA-binding protein that plays an essential role at replication centers as well as stalled replication forks in the HR repair system. Depletion of RPA leads to persistent unrepaired DSBs [
The breast cancer susceptibility genes (BRCA1 and BRCA2) and Rad51 colocalize to sites of DNA damage and have a role in both the detection and repair of DSBs [
In addition to the NHEJ and HR pathways, cells possess the DNA damage response (DDR) which functions as a cellular defense against the accumulation of genetic mutations associated with cancer progression. The DDR safeguards the genomic integrity of cells [
Regulating the phosphorylation of proteins necessary for DNA repair is key to maintaining proper repair mechanisms [
Phosphorylation events in DDR. Key factors in the DDR regulate via a series of phosphorylation events. Members of the PIKK family (ATM, ATR, and DNA-PK) are first to the DNA damage site and phosphorylate histone H2AX. As previously mentioned, DNA-PK is also a critical kinase in the NHEJ pathway. Phosphorylated H2AX recruits and phosphorylates Mediator of Damage Checkpoint protein 1 (MDC1). Once phosphorylated MDC1 then serves as a platform for further recruitment of DDR factors, including the MRN complex and RNF8. RNF8 then ubiquitinates histones and downstream recruits BRCA1, 53BP1, and RAP80.
The PIKKs, ATM and ATR, play a crucial role in DDR by relaying and amplifying the DSB damage signal. In response to DSBs, both ATM and ATR phosphorylate a multitude of substrates, including p53, and the checkpoint kinases, CHEK1 and CHEK2. These phosphorylated substrates promote cell cycle arrest and initiate DNA repair [
The activation of the protein kinase ATM initiates phosphorylation of DSB repair and cell cycle control proteins. Initial activation of the ATM kinase remains elusive. However, efficient activation requires the MRN complex and the autophosphorylation of specific ATM serine and threonine residues. ATM is recruited by the MRN complex via an interaction between ATM and the C-terminus of NBS1, a component of the MRN complex [
Another PIKK, the ATR kinase, phosphorylates ATR substrates in order to inhibit DNA replication and promote DNA repair. Activation of ATR in the DDR requires the recruitment of ATR to RPA-coated ssDNA breaks via an interacting partner, ATR-interacting protein (ATRIP). Disruption of the ATR-ATRIP complex prevents recruitment to the sites of the DSB and leads to defects in repair [
In response to DNA damage, the ATM and ATR kinases activate DSB repair enzymes via phosphorylation. ATM and ATR phosphorylates DDR proteins which participate in repair of DSBs and cell cycle control [
A crucial regulator of the DDR is histone variant H2AX, a member of the H2A family of histones, which packages and organizes DNA into chromatin. H2AX is at the center of cellular responses to DNA DSBs. In response to DNA damage, H2AX is phosphorylated on a conserved serine residue at the carboxyl terminus by PIKK family, including ATM, ATR, and DNA-PKcs. Phosphorylation of H2AX recruits DDR proteins to regions of damaged DNA, leading to delays in the cell cycle and/or DNA repair [
Another DDR factor, Mediator of Damage Checkpoint protein 1 (MDC1), is a large adaptor protein required for IRIF formation and binds
Together, the reversible phosphorylation of DNA repair factors provides a foundation for a more complete understanding of the role of molecular switches in DNA repair. Regulation of repair proteins through posttranslational modification, such as phosphorylation, provides cells with a mechanism for managing DNA repair processes. NHEJ, HR, and DDR operate together to repair damaged DNA and utilize phosphodependent binding to begin repair, recruit factors to the damage sites, and initiate signaling cascades.