The DNA damage response (DDR) is a signal transduction pathway that senses DNA damage and replication stress and sets in motion an organized response to protect the cell. A complex network of proteins is activated on induction of DNA damage. In a generalized view, sensor proteins recognize lesions and localize to sites of DNA damage. These in turn activate and recruit transducer proteins to the sites, which phosphorylate effectors or mediator proteins that influence cell activity. There are multiple cellular responses to DDR activation. One of them is cell cycle arrest, allowing time for DNA repair to occur, thereby preventing genome duplication or cell division in the presence of damaged DNA. In addition, the DNA damage response may induce cell death via apoptosis or cause cellular senescence.
DNA Damage Response signaling
There is a network of interacting pathways that mediate the cellular response to DNA damage, known as the DNA damage response (DDR). After breaks, four main sensors that can detect DSBs: PARP, Ku70/Ku80, MRN, and RPA1. Three central kinases mediate the DDR: DNA-PKcs, ATM, and ATR, members of the phosphatidylinositol-3-kinase-related kinase (PIKK) family. DNA-PKcs primarily targets proteins involved in NHEJ and is recruited through Ku70/Ku80. ATM and ATR also activate more phosphorylation through the CHK1 and CHK2 kinases. ATM is primarily activated by DSBs through the MRN complex, while ATR responds to a broader spectrum of damage including DSBs through RPA-ssDNA. PARP is thought to promote NHEJ, to mediate the accumulation of MRN, and to facilitate ATM activation. These kinases have overlapping roles in the DDR, and coordinate the response, acting to regulate DNA repair enzymes through post-translational modifications (PTMs), modifying chromatin around the damage and in the nucleus or cell to allow for repair.
The DDR can activate cell cycle checkpoints, including the G1/S, intra-S, G2/M checkpoints. Detection of DNA damage during these points in the cell cycle will prevent cell cycle progression while the damage is repaired, through the PIKKs. Checkpoints regulate DNA replication at the initiation, fork progression, and fork stability steps, and also involve the decision between cell cycle arrest, apoptosis, or senescence, through post-translational modification signaling and transcriptional regulation.
The PIKKs have several mechanisms of dealing with DNA damage. ATR activation occurs through recruitment to RPA. RPA bound to ssDNA at damage sites and replication stress centers recruits ATRIP, which recruits ATR. RPA also recruits RAD, which loads the Rad9-Hus1-Rad1 complex (9-1-1 complex); the 9-1-1 complex then loads TopBP1, which activates ATR. Activated ATR activates Chk1, which can inhibit new replication origin firing to allow for repair. ATR also directly targets replication and recombination proteins, which are necessary for fork restart, to regulate checkpoints. Another mechanism proposed is that prevention of origin firing prevents “exhaustion” of nuclear RPA levels—increasing amounts of ssDNA would occur if dormant origins fired during replication stress, and ssDNA unbound by RPA due to RPA exhaustion would be converted to DSBs. ATM may also function in the response to replication stress by activating the HR pathway, though some models suggest that ATM is only important in replication stress once DSBs are formed. Another important protein in the DDR, p53, is regulated by ATM and CHK2 in response to DSBs. P53 induces cell cycle arrest, apoptosis, or senescence in response to DNA damage through transcriptional regulation.
A major hallmark of damage is phosphorylation of histone H2AX at S139, which spreads megabases in mammals flanking the DSB; this phosphorylated form is referred to as γH2AX. Phosphorylation of H2AX is likely through ATM, and recruits MDC1. Phosphorylated MDC1 then recruits the E3 ligase RNF8, which is mediated through the RNF8 FHA domain. RNF8 with UBC13 then ubiquitinates histones H2A and H2AX. RNF168, which is also an E3 ligase, is then recruited and with UBC13 amplifies the ubiquitin signal via lysine 63 linked chains of ubiquitin on H2A and H2AX. These polyubiquitinated histones recruit RAP80 through its ubiquitin interacting motif, and RAP80 then recruits BRCA1 through Abraxas. 53BP1 is also recruited through MDC1 and RNF168, and binds dimethylated histone H4 (H4K20me2). Besides ubiquitination signaling, sumoylation by PIAS1 and PIAS4 is also important for assembly of BRCA1, 53BP1, and RNF168, at damage sites. After this signaling pathway leads to recruitment of BRCA1 and 53BP1, these factors compete and coordinate to determine which DNA repair pathway is used to repair the break; this will be discussed in more detail later. If resection is activated, it is thought to induce an ATM to ATR switch, as the ssDNA leads to ATR activation while ATM activity is attenuated. ATR then activates Chk1, which phosphorylates RAD51 and promotes repair of break through HR after DSBs at stalled forks
Besides the repair of DSBs, cells have other pathways to repair simpler damage to DNA. Mismatch Repair (MMR) and base excision repair (BER) can repair mismatches and small chemical damage, involving excision of the damaged base. Nucleotide excision repair (NER) removes more complex, bulky lesions, like pyrimidine dimers, and removes a stretch of oligonucleotides around the damage around 30 nucleotides. Intrastrand-crosslink repair (ICL) occurs by the Fanconi Anemia pathway, which requires many HR factors.