UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12

The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication. A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant. Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant. The strain showed no Weigle reactivation. From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant. It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.     

Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae.

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.     

Properties of uvrE mutants of Escherichia coli K12. I. Effects of UV irradiation on DNA metabolism

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet (UV) radiation.In an attempt to determine the underlying molecular basis for the UV sensitivity, we have compared a mutant and an isogenic wild type strain with regard to several metabolic responses to 254-nm radiation. The introduction of single-strand breaks into intracellular DNA after irradiation is normal. However, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain.These data suggest that the uvrE gene product may be involved in a reaction between the incision and excision steps in the excision repair process.     

Effects of hyperthermia on radiation-induced chromosome breakage and loss in excision repair deficient Drosophila melanogaster

Hyperthermia increased radiosensitivity with respect to gamma-ray induced chromosome loss and breakage in all stages of spermatogenesis in the wild type Oregon R strain of Drosophila melanogaster, whereas hyperthermia increased radiosensitivity to a lesser extent in cn mus (2) 201D1, an excision repair mutant with 0 per cent excision capacity and in mus (3) 308D1, a strain with 24 per cent excision capacity. The differences in hyperthermia-induced radiation sensitivity between the excision repair mutants and the wild strain may be due to the hyperthermia affecting the excision repair mechanism, suggesting that one of the possible mechanisms involved in hyperthermia-increased radiosensitivity is an effect on excision repair.     

The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.     

Defective excision and postreplication repair of UV-damaged DNA in a recL mutant strain of E. coli K-12.

Summary The mutation recL152 leads to a reduction of excision repair as measured by an increase in the time required to close uvrA uvrB dependent incision breaks, and by a reduction of host cell reactivation ability. Postreplication repair is also delayed when measured in a uvrB5 recL152 double mutant. Such a determination could not be made using the recL152 single mutant because the excision defect led to an accumulation of breaks in the unlabeled high molecular weight DNA to which the labeled DNA synthesized after irradiation must attach in order to achieve normal high molecular weight. Further, the recL gene product seems to be required to rejoin breaks in parental strand DNA which are generated during postreplication repair, since such gaps accumulate in a recL152 uvrB5 double mutant but not in a recL ⁺ uvrB5 single mutant. We have noticed a striking phenotypic similarity between recL152 and polA1 and suggest that recL152 is required for full in vivo activity of DNA polymerase I.     

The DNA translocase RAD5A acts independently of the other main DNA repair pathways,and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error‐free branch of post‐replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication‐associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single‐strand break repair (AtPARP1), as well as microhomology‐mediated double‐strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM‐mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

Effect of the uvrD mutation on excision repair.

A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene produce affects the conformation of incised deoxyribonucleic acid molecules.     

The Brucella abortus xthA-1 gene product participates in base excision repair and resistance to oxidative killing but is not required for wild-type virulence in the mouse model

Exonuclease III, encoded by the xthA gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with Escherichia coli xthA mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic xthA-1 mutant (designated CAM220) derived from virulent Brucella abortus 2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic xthA-1 mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The B. abortus xthA-1 mutant also displayed reduced resistance to killing by H2O2 and the ONOO(-)-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the xthA-1 gene product participates in protecting B. abortus 2308 from oxidative damage. Introducing a plasmid-borne copy of the parental xthA-1 gene into CAM220 restored wild-type resistance of this mutant to MMS, H2O2, and SIN-1. Although the B. abortus xthA-1 mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in C57BL/6 [corrected] mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the xthA-1 gene product participates in base excision repair and resistance to oxidative killing in B. abortus 2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.     

Bromodeoxyuridine sensitization of the ultraviolet-sensitive Escherichia coli ras- mutant to ultraviolet irradiation

Summary The Escherichia coli ras ^- mutant was sensitized to UV by bromodeoxyuridine. The extent of sensitization indicates that the ras ^- mutation probably increases UV-sensitivity by an effect on excision repair. This effect probably is uncontrolled degradation without concomitant resynthesis in a small proportion of the repair events.     

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived ‘MgPar’ donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10^?4 events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption‐deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C–MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.     

The involvement of polynucleotide ligase in the repair of UV-induced DNA damage in Escherichia coli K-12 cells.

Summary The effect of the ligts-7 mutation on cell survival and the extent of DNA repair after UV (254 nm) irradiation was determined for wild-type and uvrB5 cells of E. coli K-12 at 30° and 42°C. At the restrictive temperature (42°C) the ligts-7 mutation resulted in (i) a decrease in the extent of repair of DNA incision breaks arising during the excision repair process, and (ii) a decrease in the extent of post-replicational repair of gaps in newly-synthesized DNA. These deficiencies in DNA repair correlated with increases in cellular sensitivity to killing by UV radiation. Thus, DNA ligase plays an important role in vivo in both the excision and post-replicational repair processes.     

Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11-q21.

We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis.     

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.     

Excision-repair capacity in Streptococcus pneumoniae: cloning and expression of a uvr-like gene

Although deficient in photoreactivation and some SOS-like functions, Streptococcus pneumoniae has the capacity to carry out excision repair when exposed to UV light. The repair ability and sensitivity to UV irradiation or treatment with chemical agents in the wild type and a UV-sensitive mutant strain indicate that UV-induced pyrimidine dimers might be repaired in pneumococcus by a system similar to the uvr-dependent system in Escherichia coli. A gene complementing the mutation conferring UV sensitivity of the mutant strain has been cloned. The coding region directs the synthesis of a polypeptide with a molecular weight of 78 kDa. The relationship with uvr-like protein in E. coli is discussed.     

Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

Summary Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant.Irradiated cells from two mutants that display no excision capacity exhibit a gradual arrest of thymidine incorporation within 20 h after the initial decline. This arrest of incorporation is not observed in mutants exhibiting only partial defects in excision repair.Recovery of the ability to synthesize nascent DNA in long segments is normal in cells from the two mutants that display no excision capacity, indicating that recovery does not depend upon the excision of pyrimidine dimers from cellular DNA. Recovery of that ability is not observed, however, in cells from one partially excision-defective mutant, two of three postreplication repair-defective mutants, two of four mutants defective in both excision and postreplication repair, and one of five repair-proficient mutagen-sensitive mutants. These results indicate that recovery of normal DNA replication in irradiated Drosophila cells depends upon the activity of several functions.Abbreviation used UV ultraviolet light — principal wavelength 254 nm