The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements

Inverted repeats (IRs) that can form a hairpin or cruciform structure are common in the human genome and may be sources of instability. An IR involving the human Alu sequence (Alu-IR) has been studied as a model of such structures in yeast. We found that an Alu-IR is a mitotic recombination hotspot requiring MRE11/RAD50/XRS2 and SAE2. Using a newly developed approach for mapping rare double-strand breaks (DSBs), we established that induction of recombination results from breaks that are terminated by hairpins. Failure of the mre11, rad50, xrs2, and sae2 mutants to process the hairpins blocks recombinational repair of the DSBs and leads to generation of chromosome inverted duplications. Our results suggest an additional role for the Mre11 complex in maintaining genome stability.     

Long inverted repeats are an at-risk motif for recombination in mammalian cells

Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation. Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.     

Defective self-reactive antibody repertoire of serum IgM in patients with hyper-IgM syndrome

We have analyzed the self-reactive repertoires of IgM and IgG Abs in the serum of 19 patients with hyper-IgM syndrome (HIM) by means of a quantitative immunoblotting technique that allows for a quantitative comparison of Ab repertoires in health and disease by multiparametric statistical analysis. Normal tissue extracts of liver, lung, stomach, and kidney were used as sources of self Ags. Extracts of Pseudomonas aeruginosa and Staphylococcus epidermidis were used as sources of nonself Ags. We demonstrate a significant bias in repertoires of reactivities of IgM of patients with HIM with self Ags. Ab repertoires of IgM toward nonself Ags did not differ, however, between patients and controls. No difference was found between IgM repertoires of untreated patients and those of patients receiving substitutive treatment with i.v. IgG. IgG in the serum of HIM patients lacked reactivity with self Ags, although it exhibited a pattern of reactivity with nonself Ags that was similar to that of IgG of healthy controls. The data demonstrate that functional CD40-CD40 ligand interactions are essential for the selection of natural self-reactive B cell repertoires.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.     

Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.     

3',5'-cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: application of the forskolin criteria

The role of adenosine 3',5'-cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10(7)-10(-4) M) of forskolin produced dose-dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP-phosphodiesterase activity with 3-isobutyl-1-methyl-xanthine (10(-4) M) resulted in significant (P less than 0.05) enhancement of the forskolin effect for all but the 10(-4) M forskolin dose. Potency evaluation as judged by half-maximal stimulation of testosterone accumulation revealed median effective doses (mean +/- SE) of 1.25 +/- 0.2 x 10(-5), 1.7 +/- 0.5 x 10(-5), and 2.5 +/- 0.4 x 10(-10) M for forskolin, N6, O2'-dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time-dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P less than 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of less than or equal to 12 hr was noted for hCG- and choleragen-stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24-hr time point. Although 10(-7) M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6-fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10(-11) or 3 x 10(-11) M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10(-4) M) and one of several testicular cell agonists [forskolin (10(-4) M), choleragen (10(-9) M) or hCG (10(-9) M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin-stimulated and forskolin-amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.