C. elegans CUL-4 prevents rereplication by promoting the nuclear export of CDC-6 via a CKI-1-dependent pathway

Genome stability requires that genomic DNA is replicated only once per cell cycle. The replication-licensing system ensures that the formation of prereplicative complexes is temporally separated from the initiation of DNA replication [1-4]. The replication-licensing factors Cdc6 and Cdt1 are required for the assembly of prereplicative complexes during G1 phase. During S phase, metazoan Cdt1 is targeted for degradation by the CUL4 ubiquitin ligase [5-8], and vertebrate Cdc6 is translocated from the nucleus to the cytoplasm [9, 10]. However, because residual vertebrate Cdc6 remains in the nucleus throughout S phase [10-13], it has been unclear whether Cdc6 translocation to the cytoplasm prevents rereplication [1, 2, 14]. The inactivation of C. elegans CUL-4 is associated with dramatic levels of DNA rereplication [5]. Here, we show that C. elegans CDC-6 is exported from the nucleus during S phase in response to the phosphorylation of multiple CDK sites. CUL-4 promotes the phosphorylation and subsequent translocation of CDC-6 via negative regulation of the CDK-inhibitor CKI-1. Rereplication can be induced by coexpression of nonexportable CDC-6 with nondegradable CDT-1, indicating that redundant regulation of CDC-6 and CDT-1 prevents rereplication. This demonstrates that CDC-6 translocation is critical for preventing rereplication and that CUL-4 independently controls both replication-licensing factors.     

Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy

The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.     

Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans

In C. elegans, DOG-1 prevents deletions that initiate in polyG/polyC tracts (G/C tracts), most likely by unwinding secondary structures that can form in G/C tracts during lagging-strand DNA synthesis. We have used the dog-1 mutant to assay the in vivo contribution of various repair genes to the maintenance of G/C tracts. Here we show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G/C tracts. Similarly, we demonstrate that the C. elegans orthologs of the homologous recombination repair genes BARD1, RAD51, and XPF and the trans-lesion synthesis polymerases poleta and polkappa contribute to the prevention of deletions in dog-1 mutants. Finally, we provide evidence that the small deletions generated in the dog-1 background are not formed through homologous recombination, nucleotide excision repair, or nonhomologous end-joining mechanisms, but appear to result from a mutagenic repair mechanism acting at G/C tracts. Our data support the hypothesis that absence of DOG-1 leads to replication fork stalling that can be repaired by deletion-free or deletion-prone mechanisms.     

Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.     

Regulation of DNA replication through Sld3-Dpb11 interaction is conserved from yeast to humans

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.     

Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) requires metabolic activation to express its carcinogenic activity. This activation leads to the formation of methylating and pyridyloxobutylating agents. To determine the possible biological effects mediated by each of these metabolic pathways we have studied the activities of model compounds that are metabolized to either a methylating or pyridyloxobutylating species. Each model compound was evaluated for its mutagenic activity in both prokaryotic and eukaryotic cell systems. The model compounds were also tested for their ability to induce asynchronous replication of viral DNA sequences. We demonstrate here that both the methylating model compound acetoxymethylmethylnitrosamine (AMMN) and the pyridyloxobutylating model compound 4-(acetoxymethyl)-1-(3-pyridyl)-1-butanone (NNKOAc) were mutagenic in strains TA98, TA100, and TA1535 but not TA102. While NNKOAc appeared to be 10 times more potent than AMMN in Salmonella, AMMN was found to be a more potent mutagen in mammalian G12 cells. Both chemicals could induce asynchronous replication of polyoma DNA sequences in rat fibroblast cells carrying an integrated copy of the polyoma virus with AMMN appearing to be more active. Measurement of DNA adduct levels suggest that the damage produced by NNKOAc was at least as active as that produced by AMMN when viewed on a per adduct basis. The possible implications of the biological activities exhibited by methylating and pyridyloxobutylating model compounds to NNK induced carcinogenesis are discussed.     

Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.     

'Smoking genes': a genetic association study

Some controversy exists on the specific genetic variants that are associated with nicotine dependence and smoking-related phenotypes. The purpose of this study was to analyse the association of smoking status and smoking-related phenotypes (included nicotine dependence) with 17 candidate genetic variants: CYP2A6*1×2, CYP2A6*2 (1799T>A) [rs1801272], CYP2A6*9 (-48T>G) [rs28399433], CYP2A6*12, CYP2A13*2 (3375C>T) [rs8192789], CYP2A13*3 (7520C>G), CYP2A13*4 (579G>A), CYP2A13*7 (578C>T) [rs72552266], CYP2B6*4 (785A>G), CYP2B6*9 (516G>T), CHRNA3 546C>T [rs578776], CHRNA5 1192G>A [rs16969968], CNR1 3764C>G [rs6928499], DRD2-ANKK1 2137G>A (Taq1A) [rs1800497], 5HTT LPR, HTR2A -1438A>G [rs6311] and OPRM1 118A>G [rs1799971]. We studied the genotypes of the aforementioned polymorphisms in a cohort of Spanish smokers (cases, N = 126) and ethnically matched never smokers (controls, N = 80). The results showed significant between-group differences for CYP2A6*2 and CYP2A6*12 (both P<0.001). Compared with carriers of variant alleles, the odds ratio (OR) for being a non-smoker in individuals with the wild-type genotype of CYP2A6*12 and DRD2-ANKK1 2137G>A (Taq1A) polymorphisms was 3.60 (95%CI: 1.75, 7.44) and 2.63 (95%CI: 1.41, 4.89) respectively. Compared with the wild-type genotype, the OR for being a non-smoker in carriers of the minor CYP2A6*2 allele was 1.80 (95%CI: 1.24, 2.65). We found a significant genotype effect (all P≤0.017) for the following smoking-related phenotypes: (i) cigarettes smoked per day and CYP2A13*3; (ii) pack years smoked and CYP2A6*2, CYP2A6*1×2, CYP2A13*7, CYP2B6*4 and DRD2-ANKK1 2137G>A (Taq1A); (iii) nicotine dependence (assessed with the Fagestrom test) and CYP2A6*9. Overall, our results suggest that genetic variants potentially involved in nicotine metabolization (mainly, CYP2A6 polymorphisms) are those showing the strongest association with smoking-related phenotypes, as opposed to genetic variants influencing the brain effects of nicotine, e.g., through nicotinic acetylcholine (CHRNA5), serotoninergic (HTR2A), opioid (OPRM1) or cannabinoid receptors (CNR1).     

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes,a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.     

Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality

Polymerase zeta (Pol zeta) is an error-prone DNA polymerase [1], which in yeast is involved in trans-lesion synthesis (TLS) and is responsible for most of the ultraviolet (UV) radiation-induced and spontaneous mutagenesis [2-4]. Pol zeta consists of three subunits: REV1, a deoxycytidyl-transferase [5]; REV7, of unclear function [6]; and REV3, the catalytic subunit. REV3 alone is sufficient to carry out TLS, but association with REV1 and REV7 enhances its activity [5, 7]. Experiments using human cells treated with UV radiation indicate also that mammalian Pol zeta is involved in TLS [7]. The peculiar mutagenic activity of Pol zeta [4,7,8] suggests a possible role in somatic hypermutation of immunoglobulin (Ig) genes [9]. Here, we report that, unlike in yeast where the REV3 gene is not essential for life [4], disruption of the mouse homologue (Rev3l) resulted in early embryonic lethality. In Rev3l(-/-) embryos, no haematopoietic cells other than erythrocytes could be identified in the yolk sac. Rev3l(-/-) haematopoietic precursors were unable to expand in vitro and no haematopoietic cells could be derived from the intraembryonic haematogenic compartment (splanchnopleura). Fibroblasts could not be derived from the Rev3l(-/-) embryos, and Rev3l(-/-) embryonic stem (ES) cells could not be obtained. This is the first evidence that an enzyme involved in TLS is critical for mammalian development.     

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.     

Formation and genotoxicity of a guanine-cytosine intrastrand cross-link lesion in vivo

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 10⁹ nucleosides per Gy, which was ~300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 10⁶ nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.     

Yeast Eap1p, an eIF4E-associated protein, has a separate function involving genetic stability

A rate-limiting step during translation initiation in eukaryotic cells involves binding of the initiation factor eIF4E to the 7-methylguanosine-containing cap of mRNAs. Overexpression of eIF4E leads to malignant transformation [1-3], and eIF4E is elevated in many human cancers [4-7]. In mammalian cells, three eIF4E-binding proteins each interact with eIF4E and inhibit its function [8-10]. In yeast, EAP1 encodes a protein that binds eIF4E and inhibits cap-dependent translation in vitro [11]. A point mutation in the canonical eIF4E-binding motif of Eap1p blocks its interaction with eIF4E [11]. Here, we characterized the genetic interactions between EAP1 and NDC1, a gene whose function is required for duplication of the spindle pole body (SPB) [12], the centrosome-equivalent organelle in yeast that functions as the centrosome. We found that the deletion of EAP1 is lethal when combined with the ndc1-1 mutation. Mutations in NDC1 or altered NDC1 gene dosage lead to genetic instability [13,14]. Yeast strains lacking EAP1 also exhibit genetic instability. We tested whether these phenotypes are due to loss of EAP1 function in regulating translation. We found that both the synthetic lethal phenotype and the genetic instability phenotypes are rescued by a mutant allele of EAP1 that is unable to bind eIF4E. Our findings suggest that Eap1p carries out an eIF4E-independent function to maintain genetic stability, most likely involving SPBs.     

In vivo bypass efficiencies and mutational signatures of the guanine oxidation products 2-aminoimidazolone and 5-guanidino-4-nitroimidazole

The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.     

Active demethylation of the paternal genome in the mouse zygote

DNA methylation is essential for the control of a number of biological mechanisms in mammals [1]. Mammalian development is accompanied by two major waves of genome-wide demethylation and remethylation: one during germ-cell development and the other after fertilisation [2] [3] [4] [5] [6] [7]. Most previous studies have suggested that the genome-wide demethylation observed after fertilisation occurs passively, that is, by the lack of maintenance methylation following DNA replication and cell division [6] [7], although one other study has reported that replication-independent demethylation may also occur during early embryogenesis [8]. Here, we report that genes that are highly methylated in sperm are rapidly demethylated in the zygote only hours after fertilisation, before the first round of DNA replication commences. By contrast, the oocyte-derived maternal alleles are unaffected by this reprogramming. They either remain methylated after fertilisation or become further methylated de novo. These results provide the first direct evidence for active demethylation of single-copy genes in the mammalian zygote and, moreover, reveal a striking asymmetry in epigenetic methylation reprogramming. Whereas paternally (sperm)-derived sequences are exposed to putative active demethylases in the oocyte cytoplasm, maternally (oocyte)-derived sequences are protected from this reaction. These results, whose generality is supported by findings of Mayer et al. [9], have important implications for the establishment of biparental genetic totipotency after fertilisation, the establishment and maintenance of genomic imprinting, and the reprogramming of somatic cells during cloning.     

Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ε

During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ? (Pols α, δ and ?) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ? (pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2-5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2-5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2-5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ?, but this possibility is belied by the observation that Msh2-Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ? during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.     

Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB-irradiated Arabidopsis leaves

Replication of Arabidopsis nuclear, mitochondrial and chloroplast DNA (ncDNA, mtDNA, cpDNA) was assayed by measuring respective changes in copies per leaf, employing quantitative PCR (QPCR) analysis with genome-specific primer pairs. All three genomes showed parallel increases during growth of cotyledons and 5th leaves in planta, maintaining approximately 13 mtDNA copies and 280 cpDNA copies per haploid nuclear genome. Detached 5th leaves, which showed good growth and DNA replication on agar plates, were irradiated at (DNA-effective) UV-B fluences of 1.3-5.0 kJ m-2 and incubated under blue (photorepair-active) plus gold light or gold light only. Under blue light, replication of all genomes after all UV fluences was approximately as efficient as replication in unirradiated leaves. UV-irradiated leaves showed little growth under gold light only; 5 kJ m-2 stopped replication of all three genomes, 2.5 kJ m-2 stopped only cpDNA replication, and 1.3 kJ m-2 only delayed cpDNA replication. Immunoassays showed that 5 kJ m-2 induced about 1.2 cyclobutane pyrimidine dimers and 0.1 [6-4]photoproducts per kbp of bulk DNA, and that both photoproducts were completely removed during 2-3 days under blue light, suggesting efficient photorepair of at least ncDNA and cpDNA. The evidence for efficient photorepair of organellar DNA contrasts with previous studies of irradiated 5-day-old seedlings, and with the apparent absence of Arabidopsis photolyases bearing transit peptides.