Redundant function of DNA 1 and 3 in alternative end-joining during immunoglobulin class switch recombination

Shahnaz Masania, Li Hana, Katheryn Meeka, and Kefei Yua,1

aDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved December 22, 2015 (received for review November 2, 2015) Nonhomologous end-joining (NHEJ) is the major DNA double- whether A-EJ is a distinct pathway, consists of multiple subpath- strand break (DSB) repair pathway in mammals and resolves the ways, or is merely an aberrant form of NHEJ with missing com- DSBs generated during both V(D)J recombination in developing ponents substituted by compatible but less efficient factors. It is lymphocytes and class switch recombination (CSR) in antigen- also unclear whether A-EJ contributes to DSB repair in NHEJ- stimulated B cells. In contrast to the absolute requirement for NHEJ proficient cells at all or is only active when NHEJ is compromised. to resolve DSBs associated with V(D)J recombination, DSBs associated Much of our understanding of mechanistic details of DSB with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced repair has derived from studies of V(D)J recombination and CSR; level) by a poorly defined alternative end-joining (A-EJ) pathway. both involving DSB intermediates (1). V(D)J recombination is ini- Deletion of DNA ligase IV (Lig4), a core component of the NHEJ tiated by the recombination-activating (RAGs) that bind and pathway, reduces CSR efficiency in a mouse B-cell line capable of cleave at specific DNA sequences flanking the V, D, and J segments robust cytokine-stimulated CSR in cell culture. Here, we report that to assemble an exon encoding the variable (antigen binding) domain CSR levels are not further reduced by deletion of either of the two −/− of the B- and T-cell receptors. CSR is initiated by activation-induced remaining DNA (Lig1 and nuclear Lig3) in Lig4 cells. We cytidine deaminase (AID) in antigen-stimulated B cells that changes conclude that in the absence of Lig4, Lig1, and Lig3 function in a the IgH constant (C) region to a different isotype. AID catalyzes redundant manner in resolving switch region DSBs during CSR. DNA cytosine deamination (converting cytosines to uracils) at switch CELL BIOLOGY regions preceding each C region (6, 7). Processing of AID-generated DNA ligase | DNA double-strand break | nonhomologous end-joining | uracils, through a mechanism still not fully characterized, leads class switch recombination | DNA repair to DSB formation. Although both processes use NHEJ to join DSBs, in cells missing any of the core components of NHEJ, NA double-strand breaks (DSBs) are one of the most severe CSR is only partially defective, whereas V(D)J recombination Dforms of DNA damage that can result from pathological is completely abolished. It has been reported that the RAG conditions such as replication stress, exposure to ionizing radi- complex holds the four broken ends in a postcleavage complex ation (IR), free radicals, or other DNA-damaging drugs or be- and directs VDJ-associated DSBs into the NHEJ pathway (8, 9). In cause of failed single-strand break repair (SSBR) (1, 2). In contrast, significant levels of CSR can occur in the absence of any developing lymphocytes, programmed DSBs are essential inter- core NHEJ factors (10, 11–14), suggesting that switch region mediates for antigen receptor rearrangements, including breaks are more accessible to alternative DSB repair pathways. V(D)J recombination and Ig heavy chain class switch recom- Regardless of how broken DNA ends are processed, at least bination (CSR) (1, 2). Homologous recombination (HR) and one DNA ligase is required to ligate the two ends. Vertebrates nonhomologous end-joining (NHEJ) are the two major pathways for have three ATP-dependent DNA ligases (Lig1, Lig3, and Lig4) DSB repair. Whereas HR is restricted to the S/G2 phase of the cell (15). Lig1 and Lig4 are conserved in all eukaryotes, whereas Lig3 cycle, NHEJ is active throughout the and is generally is only present in vertebrates (15). Lig4 is a core component of considered the major pathway for DSB repair in mammals (1, 2). the NHEJ pathway and functions exclusively in NHEJ. Cells The NHEJ pathway has been extensively studied. The core components include the Ku70/Ku86 heterodimer, DNA-dependent Significance protein kinase, X-ray cross complementation factor 4 (XRCC4), and DNA ligase IV (Lig4) (1, 2). Additional NHEJ factors include the Artemis nuclease, XRCC4-like factor (XLF) (or Cernunnos), DNA ligase IV (Lig4) is essential for nonhomologous end-join- Paralog of XRCC4 and XLF, and Polymerases μ and λ.Missing ing (NHEJ), the major pathway for repairing DNA double- any of these factors results in various degrees of DSB repair deficits strand breaks in mammalian cells. An ill-defined alternative end-joining (A-EJ) pathway can also mediate end-joining in that are highly context-dependent. In general, cells lacking core cells deficient in NHEJ, although A-EJ is kinetically slower and components of NHEJ are hypersensitive to IR and abolished for V less accurate than NHEJ. Here, we report that either Lig1 or (D)J recombination but are only partially defective for CSR and Lig3 can mediate alternative end-joining in Lig4 knockout cells. competent for circulation of transfected linearized plasmids, sug- “ ” Cells having only one ligase (Lig1 or nuclear Lig3) are capable gesting that there exists an alternative way to join at least some of DNA replication and DNA repair after exposure to a wide types of DSBs. This alternative end-joining (A-EJ) pathway has range of genotoxins. These results demonstrate the remarkable recently become a focal area of research because of its implications (and unexpected) plasticity of DNA ligases in mammalian cells. in oncogenic chromosomal translocations (3), which are rare in NHEJ-proficient cells but much more frequent when NHEJ is Author contributions: K.Y. designed research; S.M. and L.H. performed research; S.M., compromised. Little is known about A-EJ other than it is kinetically L.H., K.M., and K.Y. analyzed data; and S.M., K.M., and K.Y. wrote the paper. slow and uses an increased level of microhomology (nucleotide The authors declare no conflict of interest. overlaps that can be assigned to either of the two DNA ends) This article is a PNAS Direct Submission. during joining (2, 4). A number of DNA repair factors, many of 1To whom correspondence should be addressed. Email: [email protected]. which are involved in SSBR, have been implicated in A-EJ (5), but This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the overall composition of A-EJ remains elusive. It is still unclear 1073/pnas.1521630113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1521630113 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 ABWT Lig1

Lig3

Lig4

-actin

− − − − − − − − Fig. 1. Generation and proliferation of Lig1 / Lig4 / cells. (A) Immunoblot of Lig1, Lig3, Lig4, and β-actin proteins in WT and Lig1 / Lig4 / cells; the asterisk − − − − − − − − indicates a nonspecific band. (B) Live cell counts of WT, Lig1 / , Lig4 / , and Lig1 / Lig4 / cells in unstimulated (-CIT) or stimulated (+CIT) cultures (CIT: anti- CD40, interleukin 4, and TGFβ1). Error bars indicate SE of three independent experiments.

deficient for Lig4, or its XRCC4, display the most that the major role of Lig1 is to join during severe phenotypes of NHEJ deficiency. In the absence of Lig4, DNA replication, and this function is mediated by an interaction A-EJ must rely on Lig1 or Lig3 (or both). It is generally accepted with the proliferating cell nuclear antigen (PCNA) (16). Lig3 is

− − − − − − − − − − − − Fig. 2. Drug sensitivity and CSR analysis of Lig1 / Lig4 / cells. (A) Sensitivity of WT, Lig1 / , Lig4 / , and Lig1 / Lig4 / cells to DNA-damaging agents. (B)CSR − − − − − − − − efficiency (percentage of IgA-positive cells) in WT, Lig1 / , Lig4 / ,andLig1 / Lig4 / cells. Error bars indicate SE of three independent experiments.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1521630113 Masani et al. Downloaded by guest on September 28, 2021 produced in somatic cells in two forms (mitochondrial and nu- was performed to target the second “ATG” codon of Lig3 (Fig. 3B). clear) via alternative translation initiation (17). It was recently It has been shown that mutating this ATG effectively eliminates shown that mitochondrial, but not nuclear, Lig3 is essential for nuclear-targeted Lig3 while preserving the mitochondrial Lig3 (17). cell viability (18, 19). Nuclear Lig3 stably interacts with the X-ray Several cell clones with in-frame deletions at this ATG on the + − + − − − complementation factor 1 (XRCC1), a scaffold protein that is “+” allele in Lig3 / and Lig3 / Lig4 / cells were obtained. essential for . For this reason, Lig3 is The absence of nuclear Lig3 in these cell clones (hereafter − − − − − − regarded as the primary DNA repair ligase for SSBR, although termed Lig3nuc / and Lig3nuc / Lig4 / , respectively) was con- Lig1 has also been implicated in DNA repair (15). firmed by cell fractionation and immunoblotting analyses (Fig. 3 − − We have previously reported the disruption Lig4 and Lig1 in a C and D). Proliferation of Lig3nuc / cells is comparable to that mouse B-cell line (CH12F3) capable of robust cytokine-induced nuc−/− −/− − − of WT cells, and proliferation of Lig3 Lig4 cells is com- / − − CSR (10). Lig4 CH12F3 cells undergo kinetically slow but sig- parable to that of Lig4 / cells (Fig. 3E), indicating that ablation nificant levels of CSR [∼50% of wild-type (WT) level at day 3] − − of nuclear Lig3 does not affect cell growth. (10). Switch junctions isolated from Lig4 / CH12F3 cells show − − − − − − − − increased microhomology and no direct joins (10). In the present DNA Repair and CSR in Lig3nuc / and Lig3nuc / Lig4 / Cells. Lig3nuc / study, we focused on determining which of two remaining ligases −/− CH12F3 cells display no hypersensitivity to a variety of DNA- (Lig1 and Lig3) is responsible for A-EJ during CSR in Lig4 cells. damaging agents (Fig. 4A), including Zeocin, MMS, cisplatin, or To that end, we disrupted Lig1 and Lig3 (nuclear) individually in − − hydroxyurea, consistent with previous studies in mouse embryonic / − − − − − − Lig4 CH12F3 cells. We found that Lig1 and Lig3 have re- stem cells (19). Interestingly, Lig3nuc / and Lig3nuc / Lig4 / cells dundant functions in DNA repair in response to a variety of DNA- are hypersensitive to a topoisomerase I inhibitor camptothecin damaging agents and during repair by A-EJ during CSR. (Fig. 4A), which induces SSBs. This is the first observed DNA repair Results − − − − Generation of a Lig1 / Lig4 / Cell Line. We have previously − − − − established Lig4 / and Lig1 / cell lines (10, 20) by somatic gene A targeting in a mouse mature B-cell line (CH12F3) capable of M1 M2 robust cytokine-inducible CSR in cell culture. There is no clear − − MLS difference between Lig1 / and WT cells with regard to cell proliferation, sensitivity to a variety of DNA-damaging agents, or M1 Enters mitochondria CELL BIOLOGY − − CSR (20). Lig4 / cells are, as expected, hypersensitive to Zeocin M (a DSB-inducing drug) (10). CSR is reduced, but not abolished, 2 Enters nucleus − − in Lig4 / cells (10). To determine whether Lig1 is responsible − − for the residual CSR activity in Lig4 / cells, we performed gene B Cas9 − − targeting of Lig1 in Lig4 / cells to generate cells that are de- M2 −/− −/− -CTCTGTACTGGCCCCTGTGCGATGGCAGA- ficient for both Lig1 and Lig4 (Lig1 Lig4 ). Disruption of gRNA PAM Lig1 was carried out by two rounds of gene targeting as pre- M viously described (20). Successful disruption of Lig1 was con- 1 Enters mitochondria firmed by Southern blot (Fig. S1) and immunoblotting (Fig. 1A). −/− −/− −/− Proliferation of Lig1 Lig4 cells is similar to that of Lig4 C WT Lig3nuc-/-Lig4-/- D WT Lig3nuc-/-Lig4-/- cells (Fig. 1B), which is slightly slower than WT cells when the cells are not stimulated, but markedly slower upon cytokine Lig1 Lig3 stimulation. These data indicate that Lig3 (the only DNA ligase −/− −/− Lig3 left in Lig1 Lig4 cells) is sufficient to support DNA repli- Lamin cation (i.e., Okazaki fragment ligation). * Lig4 DNA Repair and CSR in Lig1−/−Lig4−/− Cells. Previously, we have − − shown that Lig1 / cells display no obvious DNA repair defects -actin in response to exposure to a variety of DNA-damaging agents E [although these cells display modest sensitivity to methyl − − − − methanesulfonate (MMS)] (20). Lig1 / Lig4 / cells are similarly − − hypersensitive to Zeocin as Lig4 / cells (Fig. 2A). However, the − − − − modest MMS-sensitivity of Lig1 / cells is exacerbated in Lig1 / − − − − − − Lig4 / cells (Fig. 2A). Lig1 / Lig4 / cells show no hypersensi- tivity to cisplatin, hydroxyurea, or camptothecin, indicating Lig3 is sufficient to repair DNA damage induced by these drugs. No − − − − additional CSR defect was observed in Lig1 / Lig4 / cells beyond − − that observed in Lig4 / cells (Fig. 2B), indicating that Lig1 is not essential for A-EJ–mediated CSR, although this result does not rule out Lig1’s participation in A-EJ. − − Fig. 3. Gene targeting of Lig3 in Lig4 / cells. (A) Mitochondrial and nuclear − − − − − − Generation of Lig3nuc / and Lig3nuc / Lig4 / Cell Lines. Disruption forms of Lig3 produced via alternative translation initiation. M1 and M2, initi- of Lig3 was initially attempted with conventional gene targeting ation methionine residues for the mitochondrial and nuclear Lig3, respectively. + − + − − − − − (Fig. S2). Only Lig3 / and Lig3 / Lig4 / cells, but no Lig3 / or MLS, mitochondrial localization signal. (B) CRISPR-mediated disruption of nu- − − − − Lig3 / Lig4 / cells, were obtained. The failure to generate Lig3-null clear ATG results in production of only mitochondrial Lig3. (C)Immunoblot of Lig1, Lig3, Lig4, and β-actin proteins from whole-cell extracts of WT and cells is consistent with recent reports showing that mitochondrial Lig3 nuc−/− −/− Lig3 Lig4 cells; the asterisk indicates a nonspecific band. (D) Immunoblot − − − − is essential for cell viability (18, 19). Thus, we designed a strategy to of Lig3 and Lamin B proteins from nuclear extracts of WT and Lig3nuc / Lig4 / − − − − − − − − selectively ablate nuclear Lig3 without disrupting mitochondrial Lig3. cells. (E) Live cell counts of WT, Lig3nuc / , Lig4 / ,andLig3nuc / Lig4 / cells in +/− +/− −/− In Lig3 or Lig3 Lig4 cells (Fig. 3 A and B), clustered regularly unstimulated (-CIT) or stimulated (+CIT) cultures (CIT: anti-CD40, interleukin 4, interspaced short palindromic repeats (CRISPR)-mediated editing and TGFβ1). Error bars indicate SE of three independent experiments.

Masani et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 − − − − − − − − − − − − Fig. 4. Drug sensitivity and CSR of Lig3nuc / Lig4 / cells. (A) Sensitivity of WT, Lig3nuc / , Lig4 / , and Lig3nuc / Lig4 / cells to DNA-damaging agents. (B)CSR − − − − − − − − in WT, Lig3nuc / , Lig4 / , and Lig3nuc / Lig4 / cells. Error bars indicate SEMs of three independent experiments.

defect associated with Lig3 deficiency after the surprising discovery Discussion by Simsek et al. that Lig3 is mostly dispensable for nuclear DNA DNA ligases play critical roles in nearly all aspects of DNA me- repair (19). In addition, whereas Lig3 deficiency alone does not − − − − tabolism, including DNA replication, repair, and recombination cause sensitivity to MMS, Lig3nuc / Lig4 / cells are hypersensitive (15). The previous established paradigm delineates Lig1 as the − − to MMS (Fig. 4A). No CSR defect was observed in Lig3nuc / cells replicative ligase, Lig3 as the SSB repair ligase, and Lig4 as the − − − − (Fig. 4B). CSR efficiency is reduced in Lig3nuc / Lig4 / cells, but to dedicated DSB repair ligase. However, recent discoveries have − − the same level as Lig4 / cells (Fig. 4B), indicating that Lig1 alone is revealed an astonishing functional overlap among the DNA ligases sufficient to support A-EJ. Therefore, Lig1 and Lig3 have re- and greatly challenged the present ascribed function for each DNA dundant function in A-EJ during CSR, and expression of either is ligase. The finding that Lig3 deficiency has no effect on DNA re- sufficient to support efficient A-EJ. pair (18, 19) and Lig1 deficiency has no effect on DNA replication (20, 21) was unexpected. Data from this study are consistent with − − − − − − − − Switch Junctions from Lig1 / Lig4 / and Lig3nuc / Lig4 / Cells. the emerging evidence that Lig1 and Lig3 are functionally re- −/− −/− nuc−/− −/− Switch junctions from Lig1 Lig4 and Lig3 Lig4 cells dundant in DNA replication and many facets of DNA repair. We − − − − were PCR-amplified from individual switched cell clones and were not able to obtain any Lig1 / Lig3 / cells, possibly because of sequenced. We have shown previously that switch junctions from a synthetic lethality between the two as has been reported in − − Lig4 / cells display increased microhomology and no direct joins chicken DT40 cells (21). It is interesting to postulate that cells must − − − − − − − − (10). Switch junctions from Lig1 / Lig4 / or Lig3nuc / Lig4 / cells have either Lig1 or Lig3 to join Okazaki fragments during DNA − − (Fig. S3) are similar to those from Lig4 / cells (increased micro- replication and perhaps to repair spontaneous DNA damages. The homology and no direct joins) (Fig. 5), indicating that either Lig1 catalytic core of each ligase is very similar, although there are or Lig3 is sufficient for microhomology-mediated end-joining substantial differences in the other domains that likely mediate during CSR. distinct protein–protein interactions and target each ligase to

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1521630113 Masani et al. Downloaded by guest on September 28, 2021 toxicity, MMS may induce more SSBs than other SSB-inducing agents (e.g., UV, free radicals, etc.) because MMS-induced damages are more DNA-specific. An excessive number of SSBs may lead to DSBs (e.g., during DNA replication) that are toxic to NHEJ-deficient cells. Materials and Methods Reagents. Lig1 antibody (18051-1-AP) was purchased from Proteintech Group. Lig1 (sc-20222) and β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Lig4 antibody was kindly provided by David Schatz, Yale University, New Haven, CT. Lig3 antibody (611876) was purchased from BD Biosciences. Oligonucleotides were purchased Sigma-Aldrich.

Cell Culture and CSR Assay. CH12F3 cells were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) FBS and 50 μM β-mercaptoethanol. For CSR assay, cells (viability, >95%) were seeded at 5 × 104 cells/mL in the presence of 1 μg/mL anti-CD40 antibody (16-0402-86; eBioscience), 5 ng/mL of IL-4 (404-ML; R&D Systems), and 0.5 ng/mL TGF-β1 (R&D Systems 240-B) and grown for 72 h. −/− −/− nuc−/− − Fig. 5. Switch Junctions from Lig1 Lig4 and Lig3 Lig4 cells. Cells were stained with a FITC-conjugated anti-mouse IgA antibody (BD Bio- Number of switch junctions with the indicated nucleotide overlap or inser- sciences 559354) and analyzed on a LSR II flow cytometer (BD Biosciences). CSR −/− −/− tions. No significant difference was noted between Lig1 Lig4 and efficiency is determined as the percentage of IgA-positive cells. − − − − Lig3nuc / Lig4 / cells based on Mann-Whitney test (P = 0.76). Gene Targeting and CRISPR. Disruption of Lig1 was carried out in Lig4−/− cells by two rounds of gene targeting with the same targeting vector used in our − − − − different pathways in normal cells. The striking functional overlap previous study (20). The resulting Lig1 / Lig4 / genotype was confirmed by between Lig1 and Lig3 raises an important question: How does a Southern blot analysis as previously described (10, 20). Gene targeting of − − “foreign” ligase cooperate with a set of very different cofactors in a Lig3 in WT and Lig4 / cells was carried out using a targeting vector con- given process? The interaction between XRCC1 and PCNA might taining two homology blocks amplified from CH12F3 genomic DNA (Fig. S2). help explain the flexibility to use either Lig1 or Lig3 in DNA Exons 5–19 were deleted on the targeted allele. Targeted mutation of the CELL BIOLOGY replication and repair. Lig1 has a PCNA-binding motif at its N initiation codon for the nuclear Lig3 was carried out by CRISPR technology μ μ terminus that is thought to be important for Lig1’srecruitmentto (26). Briefly, 1 g of hCas9 vector (no. 41815; Addgene) and 1 g of modified the replication fork (16). In the absence of Lig1, Lig3 may access gRNA vector (backbone from Addgene no. 41824) were cotransfected into 2 × 106 cells using Amaxa nucleofector (kit V, program K-005). Transfected replication forks by an indirect interaction with PCNA bridged by cells were allowed to recover for 48 h before seeding into single cells per well XRCC1 (22). Likewise, it is conceivable that in the absence of Lig3, in a 96-well plate by limited dilution. Cell clones were picked and analyzed Lig1 may access DNA damage via an indirect interaction with by PCR, followed by Sanger sequencing of the PCR products to confirm the XRCC1 via PCNA. insertions/deletions generated by CRISPR. Our primary interest in this study is to determine which ligase (Lig1 or Lig3) is responsible for joining switch region breaks in Drug Sensitivity and MTT Assay. Cells were seeded at 1 × 105 cells/mL/well in a the absence of Lig4. We have shown previously that deletion of 24-well plate, and various DNA-damaging agents were added at different con- − − XRCC1 in Lig4 / cells does not further reduce CSR (23), sug- centrations. After 48 h of growth, cell viability was determined by a colorimetric gesting that A-EJ of switch region breaks is XRCC1-independent assay that measures the metabolic conversion of thiazolyl blue tetrazolium and not a simple form of XRCC1/Lig3-mediated SSB ligation. bromide (MTT) in the mitochondria of living cells, as described previously (10). Now, we have generated a complete panel of viable ligase- − − − − − − − − − − × 5 deficient cell strains (Lig1 / , Lig3nuc / , Lig4 / , Lig1 / Lig4 / , Cell Proliferation Analysis. Cells were seeded at 1 10 cells/mL/well in a 24- − − − − μ and Lig3nuc / Lig4 / ). Because neither Lig1 nor Lig3 deficiency well plate in regular growth medium or in the presence of 1 g/mL anti- −/− CD40 antibody (16-0402-86; eBioscience), 5 ng/mL of IL-4 (404-ML; R&D further reduces CSR efficiency in Lig4 cells, we argue that Lig1 Systems), and 0.5 ng/mL TGF-β1 (240-B; R&D Systems). Viable cells were and Lig3 are functionally redundant in A-EJ. However, it remains counted every 24 h. unclear whether Lig1 or Lig3 can be incorporated into the NHEJ machinery (albeit as lesser substitutes) for Lig4, as reviewed by Switch Junction Analysis. Individual IgA-positive clones were isolated by Pannunzio et al. (24), or whether these function in a limiting dilutions of cytokine-stimulated cultures in 96-well plates. Switch separate (parallel) pathway. Removing additional NHEJ factors junctions were amplified from individual IgA+ clones with primers KY761 (e.g., XRCC4, XLF, etc.) or expressing a catalytically inactive Lig4 5′-AACTCTCCAGCCACAGTAATGACC-3′ and KY743 5′-GAGCTCGTGGGAGTGT- in the double ligase-deficient cells may provide clues to this question. CAGTG-3′, as previously described (10). PCR products were sequenced at Mo- − − − − − − − − It is interesting that Lig1 / Lig4 / and Lig3nuc / Lig4 / cells lecular Cloning Laboratories. are hypersensitive to MMS even though any single ligase de- Cell Fractionation/Nuclear Isolation. Ten million cells were washed with 10 mL ficiency only displays a mild sensitivity to this DNA-damaging × agent. MMS is a DNA base modifier that most frequently of ice cold 1 PBS and suspended in 1 mL of ice cold lysis buffer [15 mM KCl, 10 mM Hepes (pH7.6), 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1% Nonidet methylates guanines at the N7 position (25). This modification is P-40). The cell suspension was incubated for 10 min on ice, and nuclei were not considered to directly affect DNA replication or transcrip- collected by centrifugation at 700 × g for 5 min at 4 °C. tion or to miscode the mRNA; instead, this modification triggers depurination, which can produce abasic sites (and subsequently ACKNOWLEDGMENTS. This work is supported by National Institutes of SSB) (25). We speculate that when used at a similar level of cell Health Grant R56 AI081817 (to K.Y.).

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