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. 2020 Apr 15;31(9):859–865. doi: 10.1091/mbc.E18-10-0668

Preserving genome integrity in human cells via DNA double-strand break repair

Ryan B Jensen a,*, Eli Rothenberg b,*
Editor: David G Drubinc
PMCID: PMC7185975  PMID: 32286930

Abstract

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.

INTRODUCTION

Genome integrity is paramount to sustaining the blueprint of life in both somatic and germ cells. Human cells rely on an intricate and highly regulated surveillance system that relentlessly scans the genome for issues that could compromise the integrity of DNA. Environmental and endogenous insults to DNA arise from a myriad of sources including free radicals from the oxygen we breathe, reactive metabolic byproducts, and other biochemical reactions in the cell. While multiple DNA repair pathways strive to correct damage, at times, these repair systems are pushed beyond their limits leading to the accumulation of mutations that result in human diseases such as cancer. DNA double-strand breaks (DSBs) pose a particularly large threat as these lesions lead to cell death if left unrepaired. In this brief review, we will focus on two extensively studied pathways utilized in human cells for the repair of DNA DSBs: nonhomologous end-joining (NHEJ) and homologous recombination (HR). These two DSB repair pathways result in dramatically different outcomes in terms of genome fidelity and may play a significant role in the underlying accumulation of mutations that lead to neoplastic transformation. HR proteins have recently been found to play an important role in DNA replication by protecting stalled forks from degradation and facilitating fork restart by processing lesions that impede the fork. These discoveries have fueled interest in understanding how PARP inhibitors (PARPi) may trap PARP1 on DNA creating a physical blockade to replication and require the action of HR proteins to repair and restart forks. As PARPi selectively kill HR-deficient tumors (e.g., BRCA-deficient tumors), understanding how DNA DSB repair proteins respond to and process DNA damage has important clinical implications.

CANONICAL PATHWAYS FOR REPAIR OF CHROMOSOMAL BREAKS

DNA DSBs are regarded as the most cytotoxic form of DNA damage and occur as a result of normal cellular processes, ionizing radiation (IR), and chemotherapeutics (Limp-Foster and Kelley, 2000; Lees-Miller and Meek, 2003; Lieber, 2010; Kelley, 2012). The cell must repair DSBs in a timely manner; failure to do so can lead to genetic disorders, aging, and cancer. In human cells, DSBs are repaired via two essential pathways linked to the cell cycle (Figure 1A, top): NHEJ and HR, or homology-directed repair (HDR) (Lieber, 2010; Frit et al., 2014). Each pathway employs a distinct set of core repair proteins forming functional repair complexes. The NHEJ pathway is the principal (and most rapid) pathway for DSB repair, is active throughout the cell cycle, and involves pairing, processing, and ligation of the broken ends. NHEJ also functions in B- and T-cell immune diversifying pathways by joining DSBs formed during V(D)J and class switch recombination (Lieber, 2010; Ramsden et al., 2010). The HDR pathway is active during DNA replication in S/G2 phase. A resection step commits the cell to repair by HDR resulting in single-stranded DNA (ssDNA) tails which can then invade the sister chromatid template to initiate the homology search, strand pairing, and exchange of genetic information (Daley and Sung, 2014).

FIGURE 1:

FIGURE 1:

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Molecular pathways of human DNA DSB repair. (A) DSB repair pathways are strongly regulated by the cell cycle, with NHEJ (left side) active throughout the cell cycle, whereas HDR (right side) acts mainly during replication and is utilized for the repair of damaged replication forks. Each pathway utilizes different repair factors and pathway choice is regulated by diverse requirements including: chromatin environment, site of damage, type of lesion, extent of damage, and proximity to ongoing replication and transcription. All of these factors influence the recruitment, retention, and exclusion of various repair and DDR factors via unknown mechanisms. Following formation of DSBs, DDR is activated by the PI3-like kinases: ATM, DNA-PK, and ATR. NHEJ (left) accumulation of 53BP1 at damage sites, binding of Ku, and formation of XLF, XRCC4, and LIG4 filament complexes facilitate end bridging and ligation. Depending on the complexity of the damage, other factors such as DNA-PKcs-Artemis and polymerases may be recruited to allow further processing. HR (right) is utilized for repair of damaged/stalled replication forks and involves recruitment of BRCA1 along with a suite of nucleases and associated protein complexes (CtIP, MRE11/RAD50/NBS1, BLM/EXOI, DNA2) to initiate resection. Following resection, ssDNA is bound by RPA. BRCA2 is essential for stabilizing RAD51 filaments and promoting strand invasion and the homology search. BRCA1 remains associated with the damage to enable further recruitment of BRCA2 via PALB2. Following the homology search further steps are required for the resolution of the resulting DNA junctions. (B) Fork remodeling and fork protection in replication stress. Current models propose topological stress (e.g., cross-linked DNA) or a lesion formed ahead of the replication fork will result in reversal of the replication fork mediated by factors such as RECQ1 and SMARCAL1. Following fork reversal, the reversed nascent DNA is likely protected by multiple HDR-associated proteins including BRCA2, RAD51, and RAD52 (note: several DNA repair factors have been omitted from the figure for clarity).

NHEJ

As depicted in Figure 1 (left middle), the core (or minimal) NHEJ complex is composed of the Ku70/Ku80 heterodimer (Ku), DNA Ligase 4 (LIG4)-XRCC4 complex (LX), and the XRCC4 Like factor (XLF). NHEJ can facilitate highly efficient repair of blunt and compatible DNA ends that do not require additional processing steps. However, the core NHEJ repair complex is modular and can recruit other repair factors to the break if processing is required (Chang et al., 2017). These factors include a nuclease complex composed of Ku, DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and Artemis, nucleases (Werner’s Syndrome [WRN], EXO1), polymerases (Polμ, TdT), and others (Lieber, 2010). Additionally, LIG4 was recently shown to direct processing choice prior to ligation (Conlin et al., 2017; Reid et al., 2017). The initial step of NHEJ repair involves the binding of Ku to DNA ends which acts as a cornerstone for the recruitment and assembly of a functional NHEJ complex (Figure 1, left). Following assembly, a dynamic pairing of the ends forms a synaptic complex, allowing for end alignment and ligation of the broken ends. While two early biochemical studies have suggested a possible role for DNA-PKcs in mediating synapsis (DeFazio et al., 2002; Weterings et al., 2003), numerous other studies have shown that efficient synapsis and joining are predominantly mediated by Ku, XLF, XRCC4, and LIG4 (Ahnesorg et al., 2006; Lu et al., 2007; Wu et al., 2007; Li et al., 2008; Akopiants et al., 2009; Hammel et al., 2010; Andres and Junop, 2011; Hammel et al., 2011; Ropars et al., 2011; Wu et al., 2011; Andres et al., 2012; Roy et al., 2012; Mahaney et al., 2013; Brouwer et al., 2016; Nemoz et al., 2018; Liu et al., 2019; Zhao et al., 2019).

Perhaps surprisingly, we and others have observed that XLF, XRCC4, and LIG4 form multimeric filaments both in vitro and in cells. These extended structures can facilitate pairing, synapsis, and alignment of the broken ends, while partial disruption of these filaments, via abrogated XLF–Ku interaction, results in repair deficit and radiation sensitivity (Ropars et al., 2011; Andres et al., 2012; Mahaney et al., 2013; Reid et al., 2015; Brouwer et al., 2016; Nemoz et al., 2018). It is important to note that despite these observations, we still have a very limited understanding of these filamentous complexes, including their structural, biochemical, and biophysical properties as well as how they form and localize in cells, their specific functional roles, and their interaction with chromatin. Further biochemical and structural studies of these complexes and the assembly and stability of the NHEJ machinery will provide new and improved strategies for targeting DSB repair and therapy resistance.

The kinase activity of the Ataxia Telangiectasia mutated (ATM) protein is crucial for the DNA damage response (DDR). Both ATM and DNA-PKcs phosphorylate several relevant factors associated with DNA damage signaling, yet the repair-specific role of DNA-PKcs is still subject to uncertainty. The previously ascribed cellular role of DNA-PKcs in NHEJ may stem from its functional requirement exemplified in V(D)J recombination. During V(D)J recombination, the DNA-PKcs-Artemis complex facilitates the opening of terminal hairpins at coding ends prior to ligation. However, some studies have demonstrated that DNA-PKcs is dispensable for joining blunt signal ends (Kulesza and Lieber, 1998). Several reports place the role of DNA-PKcs and Artemis at the repair of complex DNA lesions; however, one recent study indicated that depletion of DNA-PKcs had no effect on the efficiency of DSB repair by NHEJ (Liu et al., 2019). These conflicting findings indicate that further studies are required to determine the contribution of DNA-PKcs to the NHEJ repair process. Consequently, the use of DNA-PK inhibitors as a means to block NHEJ repair must be carefully reexamined in both research and clinical applications (Brandsma et al., 2017).

HR AND COMPLEX DNA LESIONS

Experimentally induced DNA DSBs derived from IR, radiomimetic chemotherapeutics, or nuclease-induced DSBs, while readily available in the laboratory setting, may not be relevant to the sources of DNA damage that ultimately result in mutations necessary for neoplastic transformation. Clearly, cancer cells are susceptible to DNA damage. The majority of our clinical efforts (chemotherapeutics, radiation) to eliminate rapidly dividing tumor cells are based on a therapeutic index that can damage the genome of a tumor cell beyond its capacity for repair while sparing normal cells. The majority of DNA DSBs in the human genome are expected to be dealt with by the NHEJ machinery. However, complex lesions resulting from cross-linking agents, topoisomerase inhibitors, PARPi, and other agents that induce topologically complex structures likely rely on HDR for eventual repair. HDR utilizes many more factors than NHEJ including nucleases (e.g., MRE11, EXO1, DNA2), recombination mediators (e.g., BRCA1, BRCA2, PALB2, RAD52), recombinase complexes (e.g., RAD51, RAD51 paralogues, RPA, RAD54), helicases, and resolvases (Delacote and Lopez, 2008; Cerbinskaite et al., 2012). The complete HDR reaction and its regulation are still subject to much uncertainty as it contains many sequential steps and subprocesses that occur over an extended period of time (6–12 h compared with 1 h for NHEJ) (Reid et al., 2015; Whelan et al., 2018), where each of these steps requires the initiation and/or completion of prior steps, and their interruption can lead to diverse repair intermediates and byproducts. The key steps (see illustration in Figure 1, right) of HDR consists of the following: 1) resection of dsDNA by nucleases, 2) loading of RAD51 onto RPA-coated ssDNA to form the nucleoprotein filament (mediated by BRCA2), 3) strand invasion and homology search, and 4) resolution of the resulting DNA structure (Holliday junctions). While NHEJ provides the most rapid form of repair throughout the cell cycle, during DNA replication, HR and NHEJ are active and can facilitate repair. Since the NHEJ pathway is reactive toward blunt dsDNA ends, whereas the HR pathway requires long ssDNA, it has been postulated that the resection of dsDNA by HR nucleases is a decisive step in pathway choice (Symington and Gautier, 2011; Shao et al., 2012; Sun et al., 2012). Experimental and clinical evidence have confirmed the importance of HDR in complex and replication-associated lesions, as patients and cell lines with HDR defects are exquisitely sensitive to cross-linking agents (e.g., cisplatin, mitomycin C) and PARPi.

DNA REPAIR AT REPLICATION FORKS

Recent studies in the DNA repair field have focused on how DNA lesions can physically impede DNA replication fork progression and the role of HDR proteins in protecting stalled or blocked forks from nucleolytic degradation. The initial idea that replication forks may reverse course after encountering damage was proposed by Hotchkiss and Higgins (Hotchkiss, 1974; Higgins et al., 1976). These reversed forks are colloquially known as “chicken foot” structures due to the three toes representing the leading, lagging, and annealed nascent DNA essentially backing up the replication fork behind the damage (Figure 1B). Fork reversal allows replication to essentially “stall” for time while the DNA repair machinery repairs the damage and prepares the fork to reinitiate replication. Zellweger et al. (2015) reported the visual evidence of these structures by electron microscopy, that is, the presence of ssDNA at regressed forks in response to genotoxic agents such as camptothecin or replication stress through hydroxyurea treatment (Zellweger et al., 2015). Further evidence demonstrated that RAD51 was present at reversed forks and may protect regressed arms from nucleolytic degradation (Hashimoto et al., 2010; Schlacher et al., 2011). Improved techniques such as iPOND (isolation of proteins on nascent DNA) and super resolution microscopy have now identified several proteins at stalled forks: SMARCAL1, ZRANB3, HLTF, RECQ1, Bloom’s Syndrome (BLM), WRN, FBH1, Fanconi Anemia (FA) proteins, and many others (Betous et al., 2013; Kolinjivadi et al., 2017; Taglialatela et al., 2017; Zadorozhny et al., 2017).

It remains unclear why so many proteins involved at different steps of DNA repair are enriched at stressed replication forks, but undoubtedly cells have a vested interest in paying close attention to molecular events that threaten genomic integrity. BRCA1 and BRCA2 have been found to play key roles at damaged replication forks either in protecting the DNA from nucleolytic degradation and/or providing an HDR-dependent path to fork restart. Considerable uncertainties exist with respect to the properties and mechanisms of fork protection-degradation and reversal processes and whether they are distinct from HDR intermediates at collapsed replication forks. Biochemical and/or superresolution microscopy-based cell biological evidence that BRCA1/2, RAD51, and related factors play a direct role in fork protection/restart remains elusive; however, these studies are eagerly anticipated.

THE BRCA PARADOX: CANCER DRIVER AND ACHILLES’ HEEL

Women who inherit a pathogenic germline mutation in BRCA2 face a serious lifetime risk of breast and ovarian cancer, whereas men face increased risk of pancreatic, prostate, and also breast cancer. BRCA2 is involved in the HDR pathway of DNA DSB repair first discovered through its interaction with RAD51, a central player in HR (Wong et al., 1997). The RAD51 protein forms a nucleoprotein filament on ssDNA and initiates a search for homology presumably in the sister chromatid (Benson et al., 1994). BRCA2 acts as a “mediator” to both bind and load RAD51 onto resected DNA DSBs (Jensen et al., 2010; Chatterjee et al., 2016). In the absence of BRCA2, RAD51 functions are compromised and HDR repair is short circuited. Loss-of-function mutations in BRCA1/2 and other HDR pathway genes (e.g., PALB2, RAD51 paralogues) can be leveraged therapeutically as they confer sensitivity to cross-linkers, platinum agents, and PARPi.

Despite BRCA2 mutation carriers possessing one wild-type copy of the allele, tumors from most patients display loss-of-heterozygosity (LOH) and are null for BRCA2 function. In some patients with germline mutations, the wild-type BRCA2 allele is retained in the tumor (Maxwell et al., 2017), and in this scenario, perhaps haploinsufficiency alone may favor tumor formation. When LOH has occurred in the tumor, the molecular ordering of events that underpin loss of the wild-type allele remains largely unknown and the genetic environment permissive for loss of BRCA2 function is an active area of investigation. This is an important point as BRCA2 knockout in mice is early embryonic lethal and knockout/knockdown in human cell culture models is usually associated with inviability (Feng and Jasin, 2017). This raises a somewhat paradoxical situation whereby BRCA2 is essential for cellular growth and viability, and yet, loss of BRCA2 can drive tumorigenesis in a specific genetic context. Furthermore, BRCA-deficient tumors are compromised for HDR repair providing a unique opportunity for therapeutic intervention with PARPi. Recent genetic studies have pointed toward a role for BRCA2 in stabilizing RAD51 at stalled replication forks and have postulated that fork protection is critical in response to PARPi (Schlacher et al., 2011; Ray Chaudhuri et al., 2016). Surprisingly, the HDR functions of BRCA2 and RAD51 appear dispensable in the replication fork protection model. Adding further to the controversy, a recent study demonstrated treatment of BRCA mutant cells results in increased replication fork speed rather than reduced velocity or fork stalling/collapse (Maya-Mendoza et al., 2018). Clearly, more work needs to be done to parse out mechanistically why BRCA proteins, and more generally the HDR pathway, is required to manage PARPi-mediated damage. To date, very few studies have utilized reconstituted biochemical assays using purified proteins and defined DNA substrates to address the mechanistic details of PARPi-dependent DNA lesions. We propose that biochemically defined systems, single molecule analysis, and superresolution visualization of these repair proteins at PARPi-induced lesions will greatly facilitate our understanding of how these clinically important molecules specifically kill HDR-deficient cells.

PARPI: THE DNA REPAIR BLOCKBUSTER DRUG

The discovery that BRCA mutant cells are selectively killed by PARPi (Bryant et al., 2005; Farmer et al., 2005) opened up a completely new therapeutic avenue for patients carrying germline BRCA mutations. Lynparza (otherwise known as Olaparib) was the first FDA-approved PARPi for the treatment of patients with BRCA-deficient ovarian cancer (Kim et al., 2015) and despite vigorous research over the past 15 years into the underlying mechanisms, we still do not know why HDR-deficient cells are selectively targeted by PARPi and how HDR-competent cells are spared. Multiple PARPi formulations have emerged from pharmaceutical companies in various stages of development from preclinical testing to FDA approval; many questions remain surrounding issues of potency and optimal treatment regimens.

PARP1 is the founding family member of a class of DNA damage sensors involved in attaching poly (ADP-ribose) PAR chains to itself (a term denoted PARylation) and target proteins at DNA lesions (Lord and Ashworth, 2017). Knockout of PARP1 and a second family member, PARP2, is individually viable in a mouse knockout; however, the double knockout PARP1–/–PARP2–/– is embryonic lethal (Menissier de Murcia et al., 2003). It is thought that PARP1 signals the presence of DNA damage, recruits various DNA repair proteins to the lesion, and eventually disengages from the DNA (through autoPARylation of itself) once appropriate DNA repair enzymes have been recruited (Lindahl et al., 1995; Pommier et al., 2016; Lord and Ashworth, 2017). The protein PARG (poly (ADP-ribose) glycohydrolase) acts as a negative regulator of PARP1 removing PARylation marks and thus, shutting down the DDR (Barkauskaite et al., 2015). The majority of PARP research has focused on PARP1, but interestingly, all clinical PARPi can inhibit both PARP1 and PARP2. The concept of “PARP trapping” whereby the PARP1 protein becomes stuck in front of a progressing replication fork has recently gained favor as the proposed mechanism of action (Murai et al., 2012, 2014). Some models suggest that prevention of catalytic PARylation underlies the synthetic lethal effects of PARPi; however, inhibition of PARylation does not seem to track with the cytotoxicity of different PARPi (Murai et al., 2012). The physical impediment of trapped PARP1 appearing as a DNA lesion may explain why removal or bypass can only be achieved using an HDR-dependent mechanism hence explaining the severe sensitivity of BRCA and other HDR-deficient cells. Whether HDR-deficient cells exposed to PARPi die by apoptosis or a mitotic catastrophe-like death from unreplicated DNA remaining following mitosis is still an open question.

GENOME INTEGRITY AND DNA REPAIR

Genome instability and dysfunctional DNA repair are linked to the etiology of severe human syndromes including developmental, immunological, and neurological disorders and cancer (McKinnon and Caldecott, 2007; Rass et al., 2007; Wilson et al., 2008; Lieber, 2010; Wu and Brosh, 2010; Wysham et al., 2012; Canugovi et al., 2013; Suhasini and Brosh, 2013; Vindigni and Gonzalo, 2013; Chang et al., 2017). Some examples of inherited genetic syndromes resulting from dysfunctional DNA damage signaling or repair include: ATM, Nijmegen Breakage Syndrome (NBS1), BLM, WRN, FA, Hereditary Nonpolyposis Colorectal Cancer (MLH1, MSH2), and hereditary breast and ovarian cancer (BRCA1, BRCA2, PALB2), among others. Genomic instability is likely the driving force that fuels neoplastic potential in the opposition of cellular factors aimed at suppressing cellular transformation. This hypothesis was elegantly proposed as the “mutator phenotype” by Larry Loeb in 1974 (Loeb et al., 1974). Accumulation of DNA damage followed by less than perfect repair offers a rich source of mutational events in critical gatekeeper pathways including tumor suppressor genes and oncogenes. In fact, these mutational events driven by DNA damage are likely the lynchpin mechanism overcoming the many barriers to transformation such as cellular proliferative capacity, angiogenesis, altered metabolic pathways, and others (Hanahan and Weinberg, 2011). Achieving an ideal balance of genetic malleability to foster tumor growth is not an easy feat and probably explains why cancer incidence is not more prevalent in the human population. Of course, age is strongly correlated with increasing cancer rates reflecting the accumulation of DNA damage over time. Continued exploration of DNA repair networks will provide an unprecedented opportunity to reveal the underlying biology of malignant transformation. Elucidation of these networks will guide the development of novel therapeutic strategies with enhanced selectivity and reduced toxicity to cancer patients.

FINAL THOUGHTS AND CONCLUSION

Despite much progress in the past few decades, we still face substantial gaps in our knowledge surrounding DNA repair mechanisms in human cells, how malfunctions lead to disease, and how DNA repair deficits can be further utilized in targeted therapy. While the first targeted DNA repair therapy—PARPi—has enjoyed its share of clinical successes for certain tumor types, resistance and relapse remain an ongoing problem. Many clever genetic and proteomic screens conducted in BRCA-mutant cell lines have identified loss-of-function genes (53BP1, REV7, Shieldin complex) that confer resistance to PARPi (Jaspers et al., 2013; Xu et al., 2015; Gupta et al., 2018; Noordermeer et al., 2018); however, clinical identification of mutations in these specific genes in actual patent tumors has remained sparse. Surprisingly, studies from breast and ovarian patient tumors resistant to cisplatin and PARPi revealed secondary reversion mutations in BRCA1 and BRCA2 back to a “wild type-like” functional state and are a potential source of bona fide resistance (Edwards et al., 2008; Sakai et al., 2008; Christie et al., 2017). Combination therapies including WEE1, ATRi, and CHK1/2 inhibitors that impair cell cycle checkpoint regulation are being tested in clinical trials with the hope they will potentiate PARPi toxicity. In order for physicians to select patients ideally suited for PARPi treatment and choose optimal combination therapies to combat resistance mechanisms, a detailed understanding of the underlying mechanism of DNA repair and PARPi is greatly needed. Another barrier hindering the progress of new therapeutic interventions is the lack of strategies for targeting NHEJ—the major DSB repair pathway. Further research focusing on the specific roles of the various structural proteins of the NHEJ machinery will provide much needed alternative approaches for targeting DNA repair (McFadden et al., 2014). Insights derived from concentrated efforts using basic experimental approaches, including biochemical, structural, and cell biology assays, along with advanced single-molecule studies have great potential to fill in these important missing details.

Abbreviations used:

ATM

Ataxia Telangiectasia mutated

BLM

Bloom’s Syndrome

DDR

DNA damage response

DNA-PKcs

DNA-dependent protein kinase catalytic subunit

DSB

double-strand break

FA

Fanconi Anemia

HDR

homology-directed repair

HR

homologous recombination

IR

ionizing radiation

LIG4

Ligase 4

LOH

loss-of-heterozygosity

NBS1

Nijmegen Breakage Syndrome

NHEJ

nonhomologous end-joining

PARPi

PARP inhibitors

ssDNA

single-stranded DNA

WRN

Werner’s Syndrome.

Footnotes

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