Selective blockage of the 3'-->5' exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3′→5′ helicase and 3′→5′ exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo­tides. In addition, we directly compared the 3′→5′ WRNp exonuclease activity with that of exo­nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo­nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo­tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.     

Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins

The RecQ helicase family comprises a conserved group of proteins implicated in several aspects of DNA metabolism. Three of the family members are defective in heritable diseases characterized by abnormal growth, premature aging, and predisposition to malignancies. These include the WRN and BLM gene products that are defective in Werner and Bloom syndromes, disorders which share many phenotypic and cellular characteristics including spontaneous genomic instability. Here, we report a physical and functional interaction between BLM and WRN. These proteins were coimmunoprecipitated from a nuclear matrix-solubilized fraction, and the purified recombinant proteins were shown to interact directly. Moreover, BLM and WRN colocalized to nuclear foci in three human cell lines. Two regions of WRN that mediate interaction with BLM were identified, and one of these was localized to the exonuclease domain of WRN. Functionally, BLM inhibited the exonuclease activity of WRN. This is the first demonstration of a physical and functional interaction between RecQ helicases. Our observation that RecQ family members interact provides new insights into the complex phenotypic manifestations resulting from the loss of these proteins.     

DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice.

This report describes an unexpected difference in the efficiency of removal of UV-induced DNA damage in the c-myc locus in splenic B lymphoblasts from two inbred strains of mice. In cells from plasmacytoma-resistant DBA/2N mice, 35% of UV-induced damage in the regulatory and 5' flank of c-myc is removed by 12 h. However, in cells from plasmacytoma-susceptible BALB/cAn mice, damage is not removed from this region. In the protein-encoding region and 3' flank of c-myc as well as in two dihydrofolate reductase gene fragments, UV damage is repaired with similar efficiency in B lymphoblasts from both strains of mice. Furthermore, in the protein-encoding portion and 3' flank of c-myc, damage is selectively removed from only the transcribed strand. No repair is detected in the nontranscribed strand. In contrast, DNA repair in the 5' flank of c-myc is not strand specific; in DNA from DBA/2N cells, UV damage is rapidly removed from both the transcribed and nontranscribed strands. In BALB/cAn cells no repair was detected in either strand in the 5'flank, consistent with the results with double-stranded, nick-translated probes to this region of c-myc. In addition to the repair studies, we have detected post-UV-damage formation: in most of the genes studied, we find that additional T4 endonuclease-sensitive sites are formed in the DNA 2 h after irradiation. Our findings provide new insights into the details of gene-specific and strand-specific DNA repair and suggest that there may be close links between DNA repair and B-cell neoplastic development.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

Effects of 1-Methylcyclopropene on Rice Growth Characteristics and Superior and Inferior Spikelet Development Under Salt Stress

Journal of Plant Growth Regulation - Salinity stress hampers rice growth and development due to its osmotic, ionic, and hormonal (ethylene) stresses. High ethylene production affects inferior and...