Interplay between DNA N-glycosylases/AP lyases at multiply damaged sites and biological consequences

Evidence has emerged that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. The first repair event occurring at a multiply damaged site (MDS) is of major importance and will largely contribute to the hazardousness of MDS. Here, using protein extracts from wild type or hOGG1-overexpressing Chinese hamster ovary cells, we investigated the initial incision rate at base damage and the formation of repair intermediates in various complex MDS. These MDS comprise a 1nt gap and 3–4 base damage, including 8-oxoguanine (oG) and 5-hydroxyuracil (hU). We report a hierarchy in base excision that mainly depends on the nature and the distribution of the damage. We also show that excision at both oG and hU, and consequently DSB formation, can be modulated by hOGG1 overexpression. Anyhow, for all the MDS analyzed, DSB formation is limited, due to impaired base excision. Interestingly, repair intermediates contain a short single-stranded region carrying a potentially mutagenic base damage. This in vitro study provides new insight into the processing of MDS and suggests that repair intermediates resulting from the processing of such MDS are rather mutagenic than toxic.     

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids,Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.IN eukaryotes, meiosis is a specialized type of cell division that produces the gametes required for sexual reproduction. In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation, termed meiosis I and II. As a result of the two divisions, four haploid cells are produced, each containing half the number of chromosomes as the diploid parent. Proper segregation at meiosis I requires connections between homologous chromosomes that are created by a combination of sister chromatid cohesion and recombination (Petronczki et al. 2003). In vegetative cells, cohesion is mediated by multisubunit ring-shaped complexes that are removed by proteolysis of the kleisin subunit, Mcd1/Scc1 (Onn et al. 2008). In meiotic cells, introduction of a meiosis-specific kleisin subunit, Rec8, allows for a two-step removal of cohesion with loss of arm cohesion at anaphase I and centromere cohesion at anaphase II (Klein et al. 1999). Missegregation of chromosomes during meiosis causes abnormal chromosome numbers in gametes that may lead to infertility and genetic disorders such as trisomy 21 or Down''s syndrome.In mitotically dividing budding yeast cells, recombination is mediated by an evolutionarily conserved RecA-like recombinase, Rad51, and occurs preferentially between sister chromatids (Kadyk and Hartwell 1992). In contrast, recombination during meiosis is initiated by the deliberate formation of double-strand breaks (DSBs) by an evolutionarily conserved, topoisomerase-like protein, Spo11, and occurs preferentially between homologous chromosomes (Jackson and Fink 1985; Schwacha and Kleckner 1997; Keeney 2001). After DSB formation, the 5′ ends on either side of the breaks are resected, resulting in 3′ single stranded (ss) tails. Rad51, and the meiosis-specific recombinase Dmc1, bind to the 3′ ssDNA tails to form protein/DNA filaments that promote strand invasion of homologous chromosomes. DNA synthesis and ligation result in the formation of double Holliday junctions, which are then preferentially resolved into crossovers (Allers and Lichten 2001; Hunter 2007).The precise roles that the Rad51 and Dmc1 recombinase activities play in meiotic recombination have been unclear because experiments have indicated both overlapping and distinct functions for the two proteins (Sheridan and Bishop 2006; Hunter 2007). While both rad51Δ and dmc1Δ mutants reduce interhomolog recombination, other studies suggest that Rad51, in complex with the accessory protein Rad54, is involved primarily in intersister DSB repair. In contrast, Dmc1, in conjunction with the accessory protein Rdh54/Tid1 (a paralog of Rad54), effects DSB repair in meiotic cells by invasion of nonsister chromatids (Dresser et al. 1997; Schwacha and Kleckner 1997; Shinohara et al. 1997a,b; Arbel et al. 1999; Bishop et al. 1999; Hayase et al. 2004; Sheridan and Bishop 2006).The preference for recombination to occur between homologous chromosomes during meiosis is created in part by Dmc1. DSBs accumulate in dmc1Δ diploids due to a failure in strand invasion (Bishop et al. 1992; Hunter and Kleckner 2001). In the efficiently sporulating SK1 strain background, these unrepaired breaks trigger the meiotic recombination checkpoint, resulting in prophase arrest (Lydall et al. 1996; Roeder and Bailis 2000). In dmc1Δ mutants, Rad51 is present at DSBs, yet there is no strand invasion of sister chromatids (Bishop 1994; Shinohara et al. 1997a). These results suggest that in addition to Dmc1 promoting interhomolog strand invasion, Rad51 activity must also be suppressed.Recent studies have shown that during meiosis Rad51 recombinase activity is inhibited by two different mechanisms that decrease the formation of Rad51/Rad54 complexes: (1) binding of the meiosis-specific Hed1 protein to Rad51, thereby excluding interaction with Rad54, and (2) reduction in the affinity of Rad54 for Rad51 due to phosphorylation of Rad54 by Mek1 (Tsubouchi and Roeder 2006; Busygina et al. 2008; Niu et al. 2009). Mek1 is a meiosis-specific kinase that is activated in response to DSBs (Niu et al. 2005, 2007; Carballo et al. 2008). In addition to phosphorylating Rad54, Mek1 phosphorylation of an as yet undetermined substrate is required to suppress Rad51/Rad54-mediated strand invasion of sister chromatids (Niu et al. 2009).To dissect the mechanism by which Mek1 suppresses meiotic intersister DSB repair, we took advantage of the ability of yeast cells to undergo haploid meiosis. The lack of homologous chromosomes in haploid cells makes it possible to examine sister-chromatid-specific events in the absence of interhomolog recombination. De Massy et al. (1994) previously observed a delay in DSB repair in haploid cells and proposed that this delay was due to a constraint in using sister chromatids. We have shown that this delay is dependent on MEK1 and utilized the haploid system to determine various biological parameters required to suppress meiotic intersister DSB repair. Our results indicate that Rad51 and Dmc1 recombinase activities have distinct roles during meiosis and that interhomolog bias is established specifically on sister chromatids through regulation of Rad51, not Dmc1. rec8Δ diploids exhibit defects in meiotic DSB repair (Klein et al. 1999; Brar et al. 2009). Given that cohesin complexes are specific for sister chromatids, we investigated the role of REC8 in intersister DSB repair and found it is required neither for suppressing intersister DSB repair during meiosis nor for the repair itself.     

The mutation frequency of 8-oxo-7,8-dihydroguanine (8-oxodG) situated in a multiply damaged site: comparison of a single and two closely opposed 8-oxodG in Escherichia coli

A multiply damaged site (MDS) is defined as > or =2 lesions within a distance of 10-15 base pairs (bp). MDS generated by ionizing radiation contain oxidative base damage, and in vitro studies have indicated that if the base damage is <3bp apart, repair of one lesion is inhibited until repair of the lesion in the opposite strand is completed. Inhibition of repair could result in an increase in the mutation frequency of the base damage. We have designed an assay to determine whether a closely opposed lesion causes an increase in adenine insertion opposite an 8-oxodG in bacteria. We have positioned the MDS (an 8-oxodG in the transcribed strand and a second 8-oxodG immediately 5' to this lesion in the non-transcribed strand) within the firefly luciferase coding region. During two rounds of replication, insertion of adenine opposite the 8-oxodG in the transcribed (T) or non-transcribed (NT) strand results in a translation termination codon at position 444 or 445, respectively. The truncated luciferase protein is inactive. We have generated double-stranded oligonucleotides that contain no damage, each single 8-oxodG or the MDS. Each double-stranded molecule was ligated into the reporter vector and the ligation products transformed into wild-type or Mut Y-deficient bacteria. The plasmid DNA was isolated and sequenced from colonies that did not express luciferase activity. In wild-type bacteria, we detected a translation stop at a frequency of 0.15% (codon 444) and 0.09% (codon 445) with a single 8-oxodG in the T or NT strand, respectively. This was enhanced approximately 3-fold when single lesions were replicated in Mut Y-deficient bacteria. Positioning an 8-oxodG in the T strand within the MDS enhanced the mutation frequency by approximately 2-fold in wild-type bacteria and 8-fold in Mut Y-deficient bacteria, while the mutation frequency of the 8-oxodG in the NT strand increased by 6-fold in Mut Y-deficient bacteria. This enhancement of mutation frequency supports the in vitro MDS studies, which demonstrated the inability of base excision repair to completely repair closely opposed lesions.     

The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae

Genomic integrity is threatened by multiple sources of DNA damage. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions and can be generated by endogenous or exogenous agents, but they can arise also during DNA replication. Sister chromatid recombination (SCR) is a key mechanism for the repair of DSBs generated during replication and it is fundamental for maintaining genomic stability. Proper repair relies on several factors, among which histone modifications play important roles in the response to DSBs. Here, we study the role of the histone H3K79 methyltransferase Dot1 in the repair by SCR of replication-dependent HO-induced DSBs, as a way to assess its function in homologous recombination. We show that Dot1, the Rad9 DNA damage checkpoint adaptor, and phosphorylation of histone H2A (γH2A) are required for efficient SCR. Moreover, we show that Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin. We propose that recruitment of Rad9 to DSB sites mediated by γH2A and H3K79 methylation contributes to DSB repair via SCR by regulating cohesin binding to damage sites. Therefore, our results contribute to an understanding of how different chromatin modifications impinge on DNA repair mechanisms, which are fundamental for maintaining genomic stability.IN eukaryotic cells, genomic integrity is ensured by the action of the DNA damage checkpoint. This checkpoint coordinates the cellular response to DNA damage, triggering cell cycle arrest and activating DNA repair mechanisms, thus providing time for the cell to repair the damage before resuming cell cycle progression (Harrison and Haber 2006). DNA double-strand breaks (DSBs) are among the most dangerous genomic lesions and, if they are not properly repaired, they can lead to fatal consequences. DSBs can be repaired either by homologous recombination (HR) or by nonhomologous end joining (NHEJ), but only HR with the sister chromatid ensures that the fidelity of genetic information is mantained. Thus, sister chromatid recombination (SCR) is the preferred mechanism of DSB repair in mitotic cells (Kadyk and Hartwell 1992; Johnson and Jasin 2000; González-Barrera et al. 2003). Since SCR occurs between identical DNA molecules, its analysis by genetic or physical methods is difficult but, recently, a physical assay to monitor the repair by SCR of a single DSB generated during replication has been developed in budding yeast (González-Barrera et al. 2003; Cortes-Ledesma and Aguilera 2006). This SCR assay is based on a circular minichromosome harboring an internal mini-HO site, which is cleaved mainly in one strand producing ∼10% DSBs during replication, in contrast to the direct and efficient DSB induction at the full-length HO site. In this way, upon HO induction, the DSB occurs only in one chromatid and the other one remains intact and available for repair (see Figure 1A). Although this assay has been used mainly to monitor unequal SCR events, it has been demonstrated that it is an accurate indicator of the proficiency in total SCR (González-Barrera et al. 2003; Cortes-Ledesma and Aguilera 2006). Using this physical assay, it has been established that Rad52, Rad59, Rad51, and Rad54, but not Rdh54/Tid1, are involved in SCR (Cortes-Ledesma et al. 2007b). Also, SMC (structural maintenance of chromosomes) proteins including the cohesin complex and the Smc5/6 complex are required for efficient SCR (Cortes-Ledesma and Aguilera 2006; De Piccoli et al. 2006; Cortes-Ledesma et al. 2007a).Open in a separate windowFigure 1.—Dot1 is required for efficient SCR. (A) Schematic of the physical assay used to monitor repair by SCR of an HO-induced DSB in the centromeric plasmid pRS316-TINV. Fragments generated after XhoI–SpeI digestion, detected by the LEU2 probe (line with asterisks) are indicated with their corresponding sizes. Since other recombination events can also lead to the 2.9-kb fragment, only the 4.7-kb band is used to measure SCR. (B) Kinetics of HO-induced DSB formation and its repair in wild-type (MKOS-3C) and dot1 (YP764) cells incubated in galactose for the indicated times. A representative Southern blot is presented showing the different fragments detected. The 3.8-kb band corresponds to the intact plasmid and equal SCR events, the 1.4-kb and 2.4-kb fragments arise after HO cut, the 2.9-kb band results from unequal SCR and IC-BIR and the 4.7-kb band is specific for unequal SCR. (C) Quantification of DSBs (1.4-kb plus 2.4-kb bands) and SCR (4.7-kb band) relative to the total DNA. Averages and standard deviations are shown. In some cases, such as the dot1 SCR values, the error bars are hidden by the graph symbols.Detection, signaling and repair of genomic lesions occur in the context of chromatin; therefore, factors regulating chromatin structure, such as histone modifications and chromatin remodelers, play important roles in the DNA damage response (Peterson and Cote 2004; Lydall and Whitehall 2005; van Attikum and Gasser 2005; Downs et al. 2007). Mec1- and Tel1-dependent phosphorylation of histone H2A at serine 129 (hereafter referred to as γH2A) is required for DSB repair by NHEJ and likely HR (Downs et al. 2000) and also mediates recruitment of cohesin to DSB sites (Unal et al. 2004). Another chromatin modification involved in the DNA damage response is the methylation of lysine 79 of histone H3 (H3K79) mediated by Dot1 (van Leeuwen et al. 2002). During meiosis, Dot1 is required for the meiotic recombination checkpoint (San-Segundo and Roeder 2000) and, in mitotic cells, Dot1-dependent H3K79 methylation is involved in the Rad9-mediated activation of the Rad53 checkpoint kinase (Giannattasio et al. 2005; Wysocki et al. 2005). Moreover, genetic analyses of the response to different DNA damaging agents, such as ionizing radiation (IR), methyl methanesulfonate (MMS), and UV, have suggested that Dot1 modulates multiple DNA repair pathways (Game et al. 2006; Toh et al. 2006; Bostelman et al. 2007; Conde and San-Segundo 2008) and also controls DSB resection (Lazzaro et al. 2008). To gain further insight in the molecular mechanisms of DNA repair impacted by Dot1 function we have used a physical assay to monitor DSB repair by SCR as a manifestation of HR repair. We provide molecular and genetic evidence indicating that Dot1, together with γH2A, promotes SCR by Rad9-mediated recruitment of cohesin to DSB sites.     

Repair of clustered uracil DNA damages in Escherichia coli

Multiply damaged sites (MDS) are defined as greater than/equal to two lesions within 10–15 bp and are generated in DNA by ionizing radiation. In vitro repair of closely opposed base damages ≥2 bp apart results in a double strand break (DSB). This work extends the in vitro studies by utilizing clusters of uracil DNA damage as model lesions to determine whether MDS are converted to DSBs in bacteria. Lesions were positioned within the firefly luciferase coding region, transformed into bacteria (wild-type, uracil DNA glycosylase-deficient, ung^–, or exonuclease III and endonuclease IV-deficient, xth^–nfo^–) and luciferase activity measured following repair. DSB formation was expected to decrease activity. Two closely opposed uracils separated by ≤7 bp decreased luciferase activity in wild-type and xth^–nfo^–, but not ung^– bacteria. Growth of bacteria to obtain plasmid-containing colonies demonstrated that the plasmid was destroyed following the mis-repair of two uracils positioned 7 bp apart. This study indicates a DSB is formed when uracil DNA glycosylase initiates repair of two closely opposed uracils ≤7 bp apart, even in the absence of the major apurinic endonucleases. This work supports the in vitro studies and demonstrates that DNA repair is not always advantageous to cells.     

Clustered DNA Lesions Containing 5-Formyluracil and AP Site: Repair via the BER System

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3''-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.     

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3′ terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.     

The mei-9a Mutant of DROSOPHILA MELANOGASTER Increases Mutagen Sensitivity and Decreases Excision Repair

The mei-9^a mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10–20 times faster than that from mei-9^a cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9^a cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.     

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.     

Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo

Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP⁺ cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.     

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H₂O₂ treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H₂O₂, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.     

Genetic Analysis of Zinc-Finger Nuclease-Induced Gene Targeting in Drosophila

Using zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved high frequencies of gene targeting in the Drosophila germline. Both local mutagenesis through nonhomologous end joining (NHEJ) and gene replacement via homologous recombination (HR) are stimulated by target cleavage. In this study we investigated the mechanisms that underlie these processes, using materials for the rosy (ry) locus. The frequency of HR dropped significantly in flies homozygous for mutations in spnA (Rad51) or okr (Rad54), two components of the invasion-mediated synthesis-dependent strand annealing (SDSA) pathway. When single-strand annealing (SSA) was also blocked by the use of a circular donor DNA, HR was completely abolished. This indicates that the majority of HR proceeds via SDSA, with a minority mediated by SSA. In flies deficient in lig4 (DNA ligase IV), a component of the major NHEJ pathway, the proportion of HR products rose significantly. This indicates that most NHEJ products are produced in a lig4-dependent process. When both spnA and lig4 were mutated and a circular donor was provided, the frequency of ry mutations was still high and no HR products were recovered. The local mutations produced in these circumstances must have arisen through an alternative, lig4-independent end-joining mechanism. These results show what repair pathways operate on double-strand breaks in this gene targeting system. They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process.EXPERIMENTAL gene targeting relies on cellular DNA repair activities. When a donor DNA carrying the desired sequence modifications is introduced into cells or organisms, successful gene replacement depends on cellular capabilities for homologous recombination (HR).We have developed a very efficient gene targeting procedure for Drosophila based on target cleavage by designed zinc-finger nucleases (ZFNs) (Bibikova et al. 2002, 2003; Beumer et al. 2006). Because the DNA-binding domain consists of Cys₂His₂ zinc fingers, these hybrid proteins are very flexible in their recognition capabilities. Each finger makes contact primarily with 3 bp of DNA, and arrays of three to four fingers provide sufficient affinity for in vivo binding. Since two ZFNs are required to cleave any single target, a pair of three-finger proteins provides adequate specificity, in principle, to attack a unique genomic sequence.When a double-strand break (DSB) is created at a specific site in the genome, DNA sequence changes result either from HR with a marked donor DNA or from inaccurate nonhomologous end joining (NHEJ). In this study we set out to determine which cellular activities support each of these processes and to learn whether the repair outcome could be biased by elimination of one or another pathway.Earlier studies showed that Drosophila uses DSB repair mechanisms that are very similar to other eukaryotic organisms (Wyman and Kanaar 2006). In the realm of HR, homologs of the Rad51 (spnA) and Rad54 (okr) proteins are required for the break-initiated meiotic recombination events needed for proper chromosome segregation in females (Kooistra et al. 1997, 1999; Ghabrial et al. 1998; Staeva-Vieira et al. 2003). Mutations in both these genes sensitize somatic cells in early developmental stages to ionizing radiation (IR) and to other DNA damaging agents. In yeast, mutations in the RAD51 gene sensitize cells to IR and lead to severe sporulation defects (Symington 2002). Mutations in RAD54 also confer sensitivity to DNA damaging agents, but are less severely affected in meiosis. In mice absence of the Rad51 protein is lethal in early embryonic development (Lim and Hasty 1996; Tsuzuki et al. 1996). Absence of Rad54 is tolerable, but confers sensitivity to IR and other agents (Essers et al. 1997).The Drosophila genome encodes components of the major NHEJ pathway, including DNA ligase IV (lig4), Xrcc4, and the Ku proteins (ku70, ku80). Loss of Lig4 sensitizes early developmental stages to ionizing radiation, and this effect is more severe in the absence of Rad54 (Gorski et al. 2003). In other assays a considerable amount of end joining still occurs in lig4 mutants (McVey et al. 2004c; Romeijn et al. 2005), suggesting a secondary or backup pathway, as has been observed in other organisms (Nussenzweig and Nussenzweig 2007). Yeasts rely more heavily on HR for DSB repair, so lig4 mutations have little effect unless HR is impaired. In contrast, lig4^−/− mice die early in embryogenesis (Barnes et al. 1998), although they can be rescued by elimination of p53 (Frank et al. 2000).The molecular process of DSB repair by HR has been studied in Drosophila by introducing a single break at a unique target either by P-element excision or by I-SceI cleavage. The evidence strongly points to an invasion and copying mechanism called synthesis-dependent strand annealing (SDSA) (see below) (Kurkulos et al. 1994; Nassif et al. 1994; McVey et al. 2004a). These events are largely dependent on spnA (McVey et al. 2004a; Johnson-Schlitz et al. 2007; Wei and Rong 2007), okr (Johnson-Schlitz et al. 2007; Wei and Rong 2007), and other factors, including mus309 (the Drosophila Bloom syndrome protein, DmBlm) (Adams et al. 2003; McVey et al. 2004b, 2007; Johnson-Schlitz and Engels 2006). When the break site is surrounded by direct repeats, repair proceeds efficiently by single-strand annealing (SSA) (Rong and Golic 2003; Preston et al. 2006).The key difference between SDSA and SSA is the mechanistic requirement for strand invasion in the former. SSA has rather modest genetic dependencies and is independent of Rad51 and Rad54, but requires that all participating molecules have ends (Symington 2002; Wyman and Kanaar 2006; Johnson-Schlitz et al. 2007; Wei and Rong 2007). In yeast, SSA is reduced in rad52 mutants, but Drosophila has no identified homolog of this gene.In this study we examined the effects of null mutations in the spnA (Rad51), okr (Rad54), and lig4 genes on ZFN-induced targeting of the Drosophila rosy (ry) locus (Beumer et al. 2006). To reveal the role of SSA, we also compared linear and circular presentation of the donor DNA.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K_d = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.     

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.     

HU protein of Escherichia coli has a role in the repair of closely opposed lesions in DNA

Closely opposed lesions form a unique class of DNA damage that is generated by ionizing radiation. Improper repair of closely opposed lesions could lead to the formation of double strand breaks that can result in increased lethality and mutagenesis. In vitro processing of closely opposed lesions was studied using double-stranded DNA containing a nick in close proximity opposite to a dihydrouracil. In this study we showed that HU protein, an Escherichia coli DNA-binding protein, has a role in the repair of closely opposed lesions. The repair of dihydrouracil is initiated by E. coli endonuclease III and processed via the base excision repair pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil by endonuclease III only when the DNA substrate contained a nick in close proximity opposite to the dihydrouracil. In contrast, HU protein did not inhibit the subsequent steps of the base excision repair pathway, namely the DNA synthesis and ligation reactions catalyzed by E. coli DNA polymerase and E. coli DNA ligase, respectively. The nick-dependent selective inhibition of endonuclease III activity by HU protein suggests that HU could play a role in reducing the formation of double strand breaks in E. coli.