DNA Fragility and Repair: Some Personal Recollections.

In this autobiographical article, I reflect on my Swedish background. Then I discuss endogenous DNA alterations and the base excision repair pathway and alternative repair strategies for some unusual DNA lesions. Endogenous DNA damage, such as loss of purine bases and cytosine deamination, is proposed as a major source of cancer-causing mutations.

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication.

Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

RAD51 protects abasic sites to prevent replication fork breakage.

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing.

Current base editors (BEs) use DNA deaminases, including cytidine deaminase in cytidine BE (CBE) or adenine deaminase in adenine BE (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for BEs capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel BEs but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

APE1-dependent base excision repair of DNA photodimers in human cells.

UV irradiation induces "bulky" DNA photodimers such as (6-4)-photoproducts and cyclobutane pyrimidine dimers that are removed by nucleotide excision repair, a complex process defective in the sunlight-sensitive and cancer-prone disease xeroderma pigmentosum. Some bacteria and lower eukaryotes can also repair photodimers by enzymatically simpler mechanisms, but such pathways have not been reported in normal human cells. Here, we have identified such a mechanism. We show that normal human cells can employ a DNA base excision repair process involving NTH1, APE1, PARP1, XRCC1, and FEN1 to rapidly remove a subset of photodimers at early times following UVC irradiation. Loss of these proteins slows the early rate of repair of photodimers in normal cells, ablates their residual repair in xeroderma pigmentosum cells, and increases UVC sensitivity ∼2-fold. These data reveal that human cells can excise photodimers using a long-patch base excision repair process that functions additively but independently of nucleotide excision repair.

HMCES protects immunoglobulin genes specifically from deletions during somatic hypermutation.

Somatic hypermutation (SHM) produces point mutations in immunoglobulin (Ig) genes in B cells when uracils created by the activation-induced deaminase are processed in a mutagenic manner by enzymes of the base excision repair (BER) and mismatch repair (MMR) pathways. Such uracil processing creates DNA strand breaks and is susceptible to the generation of deleterious deletions. Here, we demonstrate that the DNA repair factor HMCES strongly suppresses deletions without significantly affecting other parameters of SHM in mouse and human B cells, thereby facilitating the production of antigen-specific antibodies. The deletion-prone repair pathway suppressed by HMCES operates downstream from the uracil glycosylase UNG and is mediated by the combined action of BER factor APE2 and MMR factors MSH2, MSH6, and EXO1. HMCES's ability to shield against deletions during SHM requires its capacity to form covalent cross-links with abasic sites, in sharp contrast to its DNA end-joining role in class switch recombination but analogous to its genome-stabilizing role during DNA replication. Our findings lead to a novel model for the protection of Ig gene integrity during SHM in which abasic site cross-linking by HMCES intercedes at a critical juncture during processing of vulnerable gapped DNA intermediates by BER and MMR enzymes.

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase ß and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase ß and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.

Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis.

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

Impact of DNA ligase 1 and IIIα interactions with APE1 and polß on the efficiency of base excision repair pathway at the downstream steps.

Base excision repair (BER) requires a tight coordination between the repair enzymes through protein-protein interactions and involves gap filling by DNA polymerase (pol) ß and subsequent nick sealing by DNA ligase (LIG) 1 or LIGIIIα at the downstream steps. Apurinic/apyrimidinic-endonuclease 1 (APE1), by its exonuclease activity, proofreads 3' mismatches incorporated by polß during BER. We previously reported that the interruptions in the functional interplay between polß and the BER ligases result in faulty repair events. Yet, how the protein interactions of LIG1 and LIGIIIα could affect the repair pathway coordination during nick sealing at the final steps remains unknown. Here, we demonstrate that LIGIIIα interacts more tightly with polß and APE1 than LIG1, and the N-terminal noncatalytic region of LIG1 as well as the catalytic core and BRCT domain of LIGIIIα mediate interactions with both proteins. Our results demonstrated less efficient nick sealing of polß nucleotide insertion products in the absence of LIGIIIα zinc-finger domain and LIG1 N-terminal region. Furthermore, we showed a coordination between APE1 and LIG1/LIGIIIα during the removal of 3' mismatches from the nick repair intermediate on which both BER ligases can seal noncanonical ends or gap repair intermediate leading to products of single deletion mutagenesis. Overall results demonstrate the importance of functional coordination from gap filling by polß coupled to nick sealing by LIG1/LIGIIIα in the presence of proofreading by APE1, which is mainly governed by protein-protein interactions and protein-DNA intermediate communications, to maintain repair efficiency at the downstream steps of the BER pathway.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

Arabidopsis DNA glycosylase MBD4L improves recovery of aged seeds.

DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.

ADP-ribosylation of histone variant H2AX promotes base excision repair.

Optimal DNA damage response is associated with ADP-ribosylation of histones. However, the underlying molecular mechanism of DNA damage-induced histone ADP-ribosylation remains elusive. Herein, using unbiased mass spectrometry, we identify that glutamate residue 141 (E141) of variant histone H2AX is ADP-ribosylated following oxidative DNA damage. In-depth studies performed with wild-type H2AX and the ADP-ribosylation-deficient E141A mutant suggest that H2AX ADP-ribosylation plays a critical role in base excision repair (BER). Mechanistically, ADP-ribosylation on E141 mediates the recruitment of Neil3 glycosylase to the sites of DNA damage for BER. Moreover, loss of this ADP-ribosylation enhances serine-139 phosphorylation of H2AX (γH2AX) upon oxidative DNA damage and erroneously causes the accumulation of DNA double-strand break (DSB) response factors. Taken together, these results reveal that H2AX ADP-ribosylation not only facilitates BER repair, but also suppresses the γH2AX-mediated DSB response.

DNA Damage in Stem Cells.

Both embryonic and adult stem cells are endowed with a superior capacity to prevent the accumulation of genetic lesions, repair them, or avoid their propagation to daughter cells, which would be particularly detrimental to the whole organism. Inducible pluripotent stem cells also display a robust DNA damage response, but the stability of their genome is often conditioned by the mutational history of the cell population of origin, which constitutes an obstacle to clinical applications. Cancer stem cells are particularly tolerant to DNA damage and fail to undergo senescence or regulated cell death upon accumulation of genetic lesions. Such a resistance contributes to the genetic drift of evolving tumors as well as to their limited sensitivity to chemo- and radiotherapy. Here, we discuss the pathophysiological and therapeutic implications of the molecular pathways through which stem cells cope with DNA damage.

A DNA repair-independent role for alkyladenine DNA glycosylase in alkylation-induced unfolded protein response.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation.

Interlocking activities of DNA polymerase ß in the base excision repair pathway.

SignificanceBase excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase ß (Polß), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polß can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polß complexes and proposed a detailed chemical mechanism for Polß's 5'-deoxyribose-5-phosphate lyase activity.

The interplay of DNA damage and repair, gene expression, and mutagenesis in mammalian cells during oxidative stress.

We investigated the interplay among oxidative DNA damage and repair, expression of genes encoding major base excision repair (BER) enzymes and bypass DNA polymerases, and mutagenesis in mammalian cells. Primary mouse embryonic fibroblasts were challenged with oxidative stress induced by methylene blue plus visible light, and formation and repair of DNA damage, changes in gene expression, and mutagenesis were determined at increasing intervals post-treatment (0 - 192 hours). Significant formation of oxidative DNA damage together with upregulation of Ogg1, Polß, and Polκ, and no changes in Mutyh and Nudt1 expression were found in treated cells. There was a distinct interconnection between Ogg1 and Polß expression and DNA damage formation and repair whereby changes in expression of these two genes were proportionate to the levels of oxidative DNA damage, once a 3-plus hour lag time passed (P < 0.05). Equally notable was the matching pattern of Polκ expression and kinetics of oxidative DNA damage and repair (P < 0.05). The DNA damage and gene expression data were remarkably consistent with mutagenicity data in the treated cells; the induced mutation spectrum is indicative of erroneous bypass of oxidized DNA bases and incorporation of oxidized deoxynucleoside triphosphates during replication of the genomic DNA. Our findings support follow-up functional studies to elucidate how oxidation of DNA bases and the nucleotide pool, overexpression of Polκ, delayed upregulation of Ogg1 and Polß, and inadequate expression of Nudt1 and Mutyh collectively affect mutagenesis consequent to oxidative stress.

Thymine DNA glycosylase is an RNA-binding protein with high selectivity for G-rich sequences.

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase ß during base excision repair.

Base excision repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase ß (Pol ß). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol ß substrate) were subsequently bound by Pol ß, the Pol ß enzyme dissociated shortly after binding in most of the observations. In the events where Pol ß binding was followed by APE1 dissociation during substrate channeling, Pol ß remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol ß during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.