The replication machinery in the diploid cell is responsible for duplicating almost six billion base pairs before each cell division. The complete and faithful replication of these bases is essential to maintain genome stability. DNA is constantly barraged with both exogenous and endogenous insults. Some of the external sources of DNA damage are environmental toxins, ionizing radiation (IR), ultraviolet radiation (UV), and chemotherapeutic drugs. Endogenous DNA damage can arise from errors in normal replication process or from reactive oxygen species created by metabolic processes in the cell. To ensure the fidelity of replication in the face of these threats to DNA, many tightly regulated signaling pathways have evolved which are responsible for repairing the DNA and halting the replication process to allow time for this repair. Pathways which arrest the progression of replication and division are termed cell cycle checkpoints. These pathways are intimately related to DNA repair pathways. There are many repair pathways which function to correct a wide spectrum of DNA lesions. If the DNA damage in a cell is extensive or if repair fails, checkpoint pathways can help to eliminate these cells by programmed cell death or senescence in order to prevent the cell from passing on mutations. Collectively, DNA repair, cell cycle checkpoints, and apoptosis are part of the DNA damage response (DDR). If the DNA damage response fails, cells can accumulate mutations which can ultimately lead to cancer.
As the DNA damage response is essential to maintain genome stability, it is not surprising that this response is generally well-conserved across many organisms. The DNA damage response begins with sensor proteins which detect DNA damage. These sensors recruit and activate transducer proteins which, in turn, amplify the damage signal. The transducer proteins activate effector proteins, and this leads to cessation of cell cycle progression and initiation of DNA repair or programmed cell death. There are also many mediators involved in the process which carry out various functions to enhance the DNA damage response.
The response to DNA damage must be rapid so that the cell cycle is halted as quickly as possible. The response must also be transient so as to allow the resumption of the cell cycle after repair. To allow for rapid signaling, the DNA damage response is largely controlled by phosphorylation, with many of the key proteins being kinases or phosphatases.
ATM and ATR: Transducers of the DNA damage signal
The key transducers of the DNA damage response are ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), members of the phosphoinositol-3-kinase-like kinase family (PIKK). ATM and ATR are large protein kinases which, in response to DNA damage, phosphorylate over 700 downstream effectors. ATM and ATR have many overlapping substrates but ATM plays a central role in the response to double strand breaks (DSBs) and ATR is essential in the response to other types of genomic stress such as UV-induced damage and stalled replication forks.
There are many types of DNA lesions and several repair pathways have evolved to respond to the different lesions. Base damages can be fixed directly by mechanisms such as direct reversal and base excision repair (BER). Interstrand crosslinks and bulky adducts can be repaired via nucleotide excision repair (NER). DSBs and interstrand crosslinks are very dangerous to cells because genetic information is lost on both DNA strands and repair of these lesions requires more complex processes involving recombination.
DSBs are the most deleterious type of DNA lesion. Unrepaired DSBs can lead to chromosomal rearrangements, enhance genome instability and cause cell death. These DSBs can be repaired by one of two mechanisms; homologous recombination (HR) or non-homologous end-joining (NHEJ). Frank double-strand breaks, such as those caused by IR, can be repaired by either pathway; HR being prevalent in S and G2-phases of the cell cycle and NHEJ predominating in G1. Replication-associated DSBs, such as those caused by collapsed replication forks, are primarily repaired by HR. In NHEJ, DNA ends are directly religated and this process can be error-prone if bases are lost at the site of the break. Homologous recombination is very accurate in that is uses a homologous template for repair synthesis.
Like DSBs, interstrand crosslinks (ICLs), are very toxic to cells. Interstrand crosslinks prevent DNA unwinding and thereby pose a block to replication. Interstrand crosslinks can be caused by chemotherapeutic agents such as mitomycin C and cisplatin. These lesions are primarily repaired by the Fanconi anemia (FA) repair pathway. There are 13 different genes that have been shown to be involved in the FA pathway and cells with mutations in these genes are hypersensitive to agents that induce ICLs. In response to damage, the FA ubiquitin ligase core complex, made up of many of the FA proteins, ubiquitinates FANCD2 and FANCI. These modified proteins localize to the site of damage and while the exact mechanism is unclear, activate repair by coordinating the actions of several repair pathways, including NER, translesion synthesis and HR.
Inhibiting the DNA Damage response as a therapeutic strategy
Effective cancer drugs preferentially kill cancer cells over normal cells. DNA damaging drugs have long been used to treat cancer because rapidly proliferating cells, such as cancer cells, are more sensitive to DNA damage than most normal tissues. However, the use of these drugs is limited because of several normal cells types which also have a high proliferation rate. In addition, tumors are often resistant to these agents. One of the ways tumors can be protected from DNA-damaging drugs is by activation of checkpoint and DNA repair pathways. The use of inhibitors of these pathways is an attractive strategy to increase the efficacy of DNA-damaging drugs in cancer.