RecA-mediated strand exchange reactions between duplex DNA molecules containing damaged bases, deletions, and insertions

RecA protein from Escherichia coli promotes homologous pairing and strand exchange between duplex DNA molecules if one is partially single-stranded. Using linear duplexes and circles with a single-stranded gap as the substrates, this reaction generates nicked circular heteroduplex DNA and linear molecules with single-stranded ends. The completion of strand exchange can be demonstrated by the production of nicked circular heteroduplex DNA detected by gel electrophoresis and autoradiography using radiolabeled linear molecules. When the effect of ultraviolet damage to the substrate DNA was tested, strand exchange was found to pass 30 or more pyrimidine dimers in each duplex. In contrast, exchanges were blocked or severely slowed by interstrand cross-links and monoadducts produced by psoralen and 360 nm light. Deletions and insertions of from 4 to 38 base pairs in the DNA substrates had little effect on the production of nicked circular heteroduplex DNA. However, those of 120 base pairs, or greater, reduced the product yield to a level below the threshold of detection. These results contrast with those obtained in related three-stranded reactions (Bianchi, M. E., and Radding, C. M. (1984) Cell 35, 511-520), in which stable heteroduplex products with 500 or 1300 unpaired bases were obtained when the insert was located within a single-stranded circular substrate.     

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.     

Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro.

Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism. The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involved in the damage-recognition and/or incision steps, of nucleotide excision repair. Here we show that the XPA regions required for the binding to the 70 and 34 kDa subunits of RPA are located in the middle and on N-terminal regions of XPA, respectively. These regions do not overlap with the ERCC1-binding region of XPA, and a ternary protein complex of RPA, XPA and ERCC1 was detected in vitro. In addition, using the surface plasmon resonance biosensor, the binding of RPA and ERCC1 to XPA was investigated. The dissociation constants (KD) of RPA and ERCC1 with XPA were 1.9 x 10(-8 )and 2.5 x 10(-7) M, respectively. Moreover, our results suggest the sequential binding of RPA and ERCC1 to XPA.     

Effect of mutant small, acid-soluble spore proteins containing cysteine or tryptophan on DNA properties in vivo and in vitro.

Two derivatives of the alpha/beta-type small acid-soluble spore protein (SASP) SspCwt have been constructed, each containing a residue potentially useful for physico-chemical analysis of protein-protein or protein-DNA interactions. In one mutant protein (SspCtrp) residue 27 (Met) was replaced by Trp; in the second (SspCcys) residue 48 (Asn) was replaced by Cys. Both mutant proteins were expressed in Bacillus subtilis spores at levels similar to those of SspCwt, and SspCcys and SspCtrp restored ultraviolet light (UV) resistance and plasmid negative supercoiling in spores lacking major alpha/beta-type SASP to levels similar to those restored by SspCwt. While the purified mutant proteins bound more weakly to DNA than SspCwt, all three had the same relative affinity for different DNAs, ie poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than pUC19, and purified SspCcys and SspCtrp gave the same pattern of DNase protected bands with pUC19 as SspCwt. Binding of SspCcys or SspCtrp to poly(dG).poly(dC) in vitro also prevented the formation of cyclobutane type cytosine dimers upon UV irradiation, as does binding of SspCwt. These data indicate that the two mutant proteins are extremely similar to SspCwt in their interaction with DNA, and thus may be useful in probing SASP-SASP and SASP-DNA interactions directly by physical or chemical techniques. Indeed, binding of SspCtrp to poly(dG).poly(dC) resulted in a 2.5-fold enhancement of the proteins Trp fluorescence.     

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.     

Hydroosmotic activities of arginine-vasopressins modified either in positions 1, 2 and 4 or at N-terminal extensions.

Vasopressin and its synthetic analogs were studied for their effect on transepithelial water flux in frog urinary bladder. As compared with AVP, 1-deamino-8-D-arginine vasopressin (dDAVP) was about 40 times less effective in stimulating osmotic water flow. The vasopressin analogs obtained by modification in positions 1 and 2 were: [1-(1-mercapto-4-tert-butylcyclohexaneacetic acid)] AVP (I); [1-(1-mercapto-4-methylcyclohexaneacetic acid)]AVP (II); [1-(1-mercapto-4-methylcyclohexaneacetic acid)-2-O-methyltyrosine]AVP (III); and those modified in position 4 were: [1-(1-mercaptocyclohexaneacetic acid)-4-arginine] AVP (IV); [1-(2-mercaptopropionic acid)-4-arginine]AVP (V). Any of the above analogs did not influence basal, but antagonized vasopressin-stimulated water flux. N-terminally extended analogs of AVP: Ala-AVP (VI); Ser-Ala-AVP (VII) and Thr-Ser-Ala-AVP (VIII) stimulated osmotic water flux to the same extent in concentration 200 times higher as that of AVP. We conclude from these studies that vasopressin analogs (I-V) competitively antagonize vasopressin-stimulated hydroosmotic activity in frog urinary bladder probably at the epithelial vasotocin V1 and/or V2 receptor site. N-terminal extension of the vasopressin molecule did not influence the capacity of AVP to induce V2 receptor-mediated action, even when used at higher concentrations.     

The role of NBS1 in DNA double strand break repair, telomere stability, and cell cycle checkpoint control

The genomes of eukaryotic cells are under continuous assault by environmental agents and endogenous metabolic byproducts. Damage induced in DNA usually leads to a cascade of cellular events, the DNA damage response. Failure of the DNA damage response can lead to development of malignancy by reducing the efficiency and fidelity of DNA repair. The NBS1 protein is a component of the MRE11/RAD50/NBS 1 complex （MRN） that plays a critical role in the cellular response to DNA damage and the maintenance of chromosomal integrity. Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome （NBS）, a hereditary disorder that imparts an increased predisposition to development of malignancy. The phenotypic characteristics of cells isolated from NBS patients point to a deficiency in the repair of DNA double strand breaks. Here, we review the current knowledge of the role of NBS1 in the DNA damage response. Emphasis is placed on the role of NBS1 in the DNA double strand repair, modulation of the DNA damage sensing and signaling, cell cycle checkpoint control and maintenance oftelomere stability.     

Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.     

Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.Key words: telomeres, base excision repair, shelterin complex, oxidative damage, LP-BER     

ST-1, a 39-kilodalton protein in Trypanosoma brucei, exhibits a dual affinity for the duplex form of the 29-base-pair subtelomeric repeat and its C-rich strand.

In our attempt to identify telomere region-binding proteins in Trypanosoma brucei, we identified ST-1, a polypeptide with novel features. ST-1 was chromatographically purified from S-100 cell extracts and was renatured from a sodium dodecyl sulfate-protein gel as a 39-kDa polypeptide. It forms a specific complex with the trypanosome telomere repeats of TTAGGG, but more significantly, it shows a higher affinity for the 29-bp subtelomere repeats of T. brucei. These 29-mer boxes are a large tandem series of telomere-derived repeats which separate the simple telomere DNA from middle-repetitive telomere-associated sequences on many chromosomes. ST-1 is the first example of a protein binding within such large repetitive subtelomere elements in trypanosomes or other organisms. ST-1 is also novel in that it has a selective affinity for the C-rich strands of both the subtelomeric 29-mer and the telomere repeats, comparable to that for the duplex form of the respective repeats. All previously described telomere-binding proteins have affinity for only the duplex form or for the G-rich strand. This C-rich strand binding specificity of ST-1 may provide insight into this protein's mechanism of binding in vivo.     

Recognition and removal of oxidized guanines in duplex DNA by the base excision repair enzymes hOGG1, yOGG1, and yOGG2

8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg. In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1. The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts. Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme. In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex. No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1. However, yOGG2 removed Sp at a faster rate than Gh. Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic. Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity. However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG. The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo. Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.     

Synergism between base excision repair,mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.     

Impact of telomerase ablation on organismal viability, aging, and tumorigenesis in mice lacking the DNA repair proteins PARP-1, Ku86, or DNA-PKcs

The DNA repair proteins poly(ADP-ribose) polymerase-1 (PARP-1), Ku86, and catalytic subunit of DNA-PK (DNA-PKcs) have been involved in telomere metabolism. To genetically dissect the impact of these activities on telomere function, as well as organismal cancer and aging, we have generated mice doubly deficient for both telomerase and any of the mentioned DNA repair proteins, PARP-1, Ku86, or DNA-PKcs. First, we show that abrogation of PARP-1 in the absence of telomerase does not affect the rate of telomere shortening, telomere capping, or organismal viability compared with single telomerase-deficient controls. Thus, PARP-1 does not have a major role in telomere metabolism, not even in the context of telomerase deficiency. In contrast, mice doubly deficient for telomerase and either Ku86 or DNA-PKcs manifest accelerated loss of organismal viability compared with single telomerase-deficient mice. Interestingly, this loss of organismal viability correlates with proliferative defects and age-related pathologies, but not with increased incidence of cancer. These results support the notion that absence of telomerase and short telomeres in combination with DNA repair deficiencies accelerate the aging process without impacting on tumorigenesis.     

The polycystin-1 C-type lectin domain binds carbohydrate in a calcium-dependent manner, and interacts with extracellular matrix proteins in vitro

Mutations in the PKD1 gene are responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). This gene encodes a large membrane associated glycoprotein, polycystin-1, which is predicted to contain a number of extracellular protein motifs, including a C-type lectin domain between amino acids 403--532. We have cloned and expressed the PKD1 C-type lectin domain, and have demonstrated that it binds carbohydrate matrices in vitro, and that Ca(2+) is required for this interaction. This domain also binds to collagens type I, II and IV in vitro. This binding is greatly enhanced in the presence of Ca(2+) and can be inhibited by soluble carbohydrates such as 2-deoxyglucose and dextran. These results suggest that polycystin-1 may be involved in protein-carbohydrate interactions in vivo. The data presented indicate that there may a direct interaction between the PKD1 gene product and an ubiquitous extracellular matrix (ECM) protein.     

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.     

Superhelical stress restrained in plasmid DNA during repair synthesis initiated by the UvrA, B and C proteins in vitro.

Purified UvrA, UvrB, UvrC, UvrD, PolA and Lig proteins from Escherichia coli have been used to assess the effect of nucleotide excision repair on the conformation of native negatively supercoiled plasmid DNA in an in vitro test system. The analysis of labeled reaction products on specific gel systems suggests that the Uvr excinuclease has the ability to restrain the superhelical stress in the template DNA during the repair process. This feature, observed in the case of the Uvr system is not found if the repair reaction is initiated by T4 endonuclease V or Micrococcus luteus UV endonuclease.