Roles of ATM and ATR in DNA double strand breaks and replication stress
Introduction
Thousands of instances of DNA damage occur in a cell on a daily basis, caused by exogenous agents including ultraviolet (UV) radiation, ionising radiation (IR) and a variety of chemical agents as well as endogenous processes including errors in DNA replication and reactive metabolites such as reactive oxygen species (ROS) (Lindahl and Barnes, 2000). Cells have therefore evolved a number of elaborate DNA damage response (DDR) pathways to counteract DNA damage, stop the build-up of damaging DNA lesions and prevent dysregulation of the cell cycle. Unsurprisingly given their key roles in maintaining genome integrity, mutations in DDR components are regularly observed in a variety of cancers and many factors have been identified as therapeutic targets (Ciccia and Elledge, 2010; Weber and Ryan, 2015).
Double-strand breaks (DSB), estimated to occur endogenously at an average rate of ∼50 DSBs per cell per day (Vilenchik and Knudson, 2003), are the most dangerous form of DNA damage. If left unrepaired, loss of cell cycle stalling in the presence of DSBs results in gross chromosomal abnormalities and ultimately cancer. The two major pathways used for DSB repair in eukaryotic cells are non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ repairs DSBs by ligation of DNA ends without the use of a template or sister chromatid (Chaplin and Blundell, 2020) which whilst significantly faster than repair by HR and able to operate throughout the cell cycle, can lead to loss of genetic information. HR is the dominant pathway for DSB repair in S phase of the cell cycle, during which genetic material is doubled in preparation for cell division. Using a template for repair of DSBs, HR is the most faithful repair pathway for DSBs.
Two proteins that are central components of the HR-mediated DSB response are the master kinases ATM (Ataxia Telangiectasia Mutated, yeast orthologue Tel1) and ATR (ATM and Rad3-Related, yeast orthologue Mec1). Conserved across eukaryotes, ATMTel1 and ATRMec1 are related kinases from the PIKK (phosphatidylinositol 3-kinase-related kinase) family, members of which also include mTOR, TRAAP, SMG-1 and DNA-PKcs, the latter of which is involved in NHEJ in vertebrates (Lempiäinen and Halazonetis, 2009; Williams et al., 2020). The PIKK family members are large kinases of up to >4000 residues with a conserved architecture; an N-terminal region containing a varying number of HEAT repeats, a FAT (FRAPP, ATM, TRAAP) domain of ∼1200 residues and a C-terminal kinase domain. Where previously there was limited structural information and therefore molecular details of how these giant kinases function, progress in Cryo-EM has recently provided a number of valuable and increasingly high-resolution structural insights into ATMTel1 (Baretić et al., 2017; Jansma et al., 2020; Xin et al., 2019; Yates et al., 2020) and ATRMec1(Rao et al., 2018; Tannous et al., 2020; Wang et al., 2017). ATMTel1 forms a heterodimer with a kinase to kinase arrangement, resembling a butterfly in appearance whilst ATRMec1 forms an obligatory heterodimer with its partner ATRIP (yeast orthologue Ddc2), which also consists of a large number of HEAT repeats (Fig. 1). The ATR-ATRIPMec1-Ddc2 heterodimer itself dimerises, forming a dimer of heterodimers with a similar butterfly-like appearance to that observed for ATM. The structures and regulations of ATMTel1 and ATR-ATRIPMec1-Ddc2, in particular key elements that contribute to autoinhibition and regulation, have been discussed in detail in a recent review (Williams et al., 2020).
ATMTel1 and ATRMec1 are recruited to and activated in response to DNA damage, ensuring proper coordination of the cell cycle during HR-mediated repair of DNA damage and preventing accumulation of dangerous chromosomal abnormalities. In addition to damage caused by breakage of the DNA in the absence of replication, damage also arises from replication stress, resulting in stalled replication forks which if left unrepaired lead to fork breakage and genomic instability. Many of the components that are central to repair of DSBs, including ATMTel1 and ATRMec1 are also key factors in protecting stalled replication forks under conditions of replicative stress. This review discusses the functions of ATMTel1 and ATRMec1 in HR-mediated DSB repair and replication stress and how their activity in response to these different forms of DNA damage are primarily governed by control of ATMTel1 and ATRMec1 localisation, allowing these two master kinases to play similar yet functionally distinct roles in essential maintenance of genome integrity.
Section snippets
ATMTel1 and HR-mediated DSB repair
Following a DSB that occurs independently of replication, for example due to exogenous DNA damage caused by UV radiation, the early stages of HR are concerned with sensing DNA ends and resection of the break site. MRN (MRX in yeast), a heterotrimeric complex consisting of MRE11, RAD50 and NBS1 (Xrs2 in yeast) is present at consistent levels in the nucleus throughout the cell cycle (Maser et al., 1997) and rapidly relocates to DSBs (Mirzoeva and Petrini, 2001), suggesting that MRN/X is
Regulation of ATMTel1 at DSBs
With ATMTel1 and ATRMec1 being responsible for linking DNA damage repair to cell cycle progression, it is critical that the activity of both kinases is strictly regulated. Accordingly, both ATMTel1 and ATRMec1 have low basal activities and require activator proteins in order to phosphorylate their substrates. Both kinases, as with the other PIKKs, are held in an autoinhibited state. For ATMTel1, recent Cryo-EM structures have identified a region near the C-terminus of the kinase domain known as
ATMTel1 substrates and downstream signalling in DSB repair
Over 700 substrates have been identified for ATMTel1 and ATRMec1 and there is significant overlap in substrate specificity with both kinases phosphorylating S/TQ motifs in their targets (Matsuoka et al., 2007). Several proteins involved in early stages of HR are also ATM substrates. Despite the vast number of identified substrates, the role of phosphorylation by ATMTel1 and ATRMec1 in many of them is still unclear. However, given their roles as master kinases, many ATMTel1 and ATRMec1
ATMTel1 in response to replication stress
Faithful replication of the genome requires the proper formation and function of every replication fork that is travelling along the DNA. Numerous replication origins are simultaneously active during DNA replication in S phase and replication stress occurs when forks are slowed or stalled for a number of reasons including endogenous DNA lesions such as crosslinks, regions of high complexity, collision of replication forks with replication machinery and metabolic deficiencies such as low dNTP
Recruitment and activation of ATR-ATRIPMec1-Ddc2 at DSBs
After resection of the DSB, the generated ssDNA is coated by RPA, the main ssDNA-binding protein in eukaryotes. The binding of RPA is a key occurrence in processing of DNA damage as well as replication, preventing secondary structure formation, protecting ssDNA from degradation by nucleases and acting as a binding platform for numerous partners (Fanning et al., 2006; Maréchal and Zou, 2015). ATR-ATRIPMec1-Ddc2 is recruited to RPA-coated ssDNA by binding RPA via the N-terminal region of ATRIPDdc2
ATRMec1 substrates and downstream signalling in DSB repair
The major pathway through which ATRMec1 inhibits cell cycle progression in response to DNA damage is via ATR-CHK1 (Mec1-Rad53 in yeast) signalling (Fig. 4). While ATM-CHK2 signalling is crucial for cell cycle arrest at the G2/M checkpoint in response to DSBs, ATRMec1-CHK1Rad53 signalling is essential for cell survival and the main effector in continuing the prevention of mitotic entry at the G2 checkpoint; inhibition of this signalling pathway leads to accumulation of DNA damage and failure to
ATRMec1 in response to replication stress
DNA replication stress including fork stalling can be detrimental to cells if unresolved. Fork reversal commonly occurs to protect and repair stalled replication forks (Neelsen and Lopes, 2015) – the new DNA strands anneal together and this appears to be a response to most replication stress, with an equilibrium between fork reversal and fork progression (Couch and Cortez, 2014; Zellweger et al., 2015). ATR-ATRIP is a key regulator in repair of DNA damage arising from replication stress because
Concluding remarks
ATMTel1 and ATRMec1 have significantly overlapping and interdependent roles in response to DSBs, coordinating end resection and recruitment of repair proteins with the checkpoint response. ATRMec1 plays integral roles in orchestrating the repair of deleterious DNA structures formed during replication stress and in DSB repair. ATRMec1 plays additional roles in replication, distinct from CHK1-associated checkpoint activation and downstream DDR. Conversely, ATMTel1 has less of a direct role in
Funding
Work in XZ’s lab is supported by the Wellcome Trust (098412/Z/12/Z, 2012–2018 and 210658/Z/18/Z, 2019-).
Data statement
This review article does not contain any new data.
Declaration of competing interest
The authors declare that they have no competing financial interests or personal relationships that could be perceived to have influenced the work reported in this paper.
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