Growth Phase-Dependent Regulation of Vsr Endonuclease May Contribute to 5-Methylcytosine Mutational Hot Spots in Escherichia coli

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of Escherichia coli is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.     

Effects of Anesthetic Ketamine on Anxiety-Like Behaviour in Rats

There is scarce information regarding the effects of anesthetic doses of the non-competitive N-methyl-d-aspartate receptor antagonist ketamine on anxiety. The current study evaluated the acute effects of intraperitoneally (i.p.) administered anesthetic ketamine (100 mg/kg) i.p. on anxiety in rats. For this purpose, the light/dark and the open field tests were utilized. The effects of anesthetic ketamine on motility were also examined using a motility cage. In the light/dark test, anesthetic ketamine, administered 24 h before testing reduced the number of transitions between the light and dark compartments and the time spent in the light compartment in the rats compared with their control cohorts. In addition, ketamine was found to exert a depressive effect on rats’ motility. In the open field test, animals treated with anesthetic ketamine 24 h before testing spent essentially no time in the central area of the apparatus, decreased horizontal ambulatory activity, and preserved to a certain extent their exploratory behaviour compared to their control counterparts. The results suggest that, in spite of its hypokinetic effect, a single anesthetic ketamine administration apparently induces an anxiety-like state, while largely preserving exploratory behaviour in the rat. These effects were time-dependent they since they were extinguished when testing was carried out 48 h after anesthetic ketamine administration.

     

The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage

The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.The Escherichia coli Dcm protein methylates the second C of CCWGG sites (W = A or T). Deamination of 5-methylcytosine converts CG base pairs to T/G mismatches, causing CCWGG-to-CTWGG transition mutations. Very-short-patch (VSP) repair minimizes these mutations (2). Repair is initiated by a sequence- and mismatch-specific endonuclease, Vsr, which cleaves the DNA 5′ of the T. DNA polymerase I removes the T along with a few 3′ nucleotides and resynthesizes the missing bases, restoring the CG base pair. Vsr is both necessary and sufficient for initiating VSP repair. However, two other proteins, MutS and MutL, enhance VSP repair of deamination damage (1).MutS and MutL are best known for their roles in postreplication mismatch repair (MMR) (9, 11). MutL couples mismatch recognition by MutS to the activation of MutH, an endonuclease that cleaves the unmethylated strand of GATC sequences that are transiently hemimethylated following DNA replication. The nicked strand, containing the erroneous base, is removed by the UvrD helicase and one of several exonucleases to beyond the mismatch and then resynthesized by DNA polymerase III.MutL stimulates the endonuclease activities of both Vsr and MutH in vitro (8, 17). The requirements for stimulation are the same: a mismatch, MutS, and ATP hydrolysis by MutL (8, 8a). Cross-linking studies showed that MutH and Vsr interact with the same region in the N-terminal domain of MutL (Heinze et al., submitted). Competition of Vsr with MutH for access to MutL explains the ability of Vsr to inactivate MMR in vivo when overexpressed (6, 13). Thus, the interactions of the two repair endonucleases with MutL are structurally and functionally very similar.In contrast to MMR, where the cleavage site for MutH may be several kilobases away from the mismatch, VSP repair requires that mismatch recognition and endonucleolytic cleavage occur at the same C(T/G)WGG site. How MutS and MutL stimulate VSP repair if MutS and Vsr compete for the same mismatch remains unknown (2, 12). We hypothesized that MutS binds the mismatch first and that a MutS-MutL complex then recruits Vsr. If so, then the MMR proteins would initially mask the mismatch, making the interaction of Vsr with MutL independent of lesion identity.To test this hypothesis, we studied the interaction of MutL with Vsr and with MutH in response to two types of mismatch by using a bacterial two-hybrid assay (10). This assay detects all known interactions among the Mut proteins: homodimerization of MutS and MutL, interaction of MutL with MutS and with MutH, and interaction of Vsr with the N-terminal domain of MutL (15). We found no false positives or false negatives. Furthermore, since the assay relies on reconstitution of a soluble protein (adenylate cyclase), the DNA repair proteins are free to interact with the DNA (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Known interactions among repair proteins as detected by the bacterial two-hybrid assay. The T18 and T25 subunits of CyaA are fused to any two repair proteins (illustrated here by MutL and Vsr), allowing measurement of all pairwise interactions as units of β-galactosidase (β-gal). T25 fusions are repair proficient. CRP, cyclic AMP (cAMP) receptor protein; P, lac operon promoter; RNAP, RNA polymerase.2-Aminopurine (2AP) mispairs with C during DNA replication, causing transition and frameshift mutations (5). The transitions are due primarily to the mismatch itself; the frameshifts are due to saturation of MMR, which leaves slipped-strand intermediates caused by DNA replication errors unrepaired (19). MutS and MutL bind to 2AP/C lesions (22), although the lesions may not be subject to MMR (19). As shown in Fig. ​Fig.2,2, treatment with 2AP causes a dose-dependent increase in the interaction of MutL with both Vsr and MutH; dimerization of MutL and interaction of MutL with MutS are somewhat increased.Open in a separate windowFIG. 2.Effect of 2AP treatment on protein-protein interactions in the bacterial two-hybrid assay. Results in units of β-galactosidase ± standard errors of the means (n = 9) are shown for BTH101(F galE15 ga1K16 rpsL1 hsdR2 mcrA1 mcrB1 cyaA-99) cells treated with 2AP as described previously (5, 19). Cells were cotransformed with pT18 and pT25 vectors (light gray bars), pT18-mutS and pT25-mutL (white bars), pT18-vsr and pT25-mutL (gray bars), pT18-mutH and pT25-mutL (black bars), or pT18-mutL and pT25-mutL (mottled bars). (NB: The dose-response curve for the pT18-mutS pT25-mutS transformants is similar to that of the pT18-mutL pT25-mutL transformants; it has been omitted for graphical clarity since the MutS-MutS interaction gives very high units of β-galactosidase activity [15]).The MutY adenine glycosylase removes A''s which have mispaired with oxidized guanine (8-oxoG) during DNA replication. Cells with a deletion of mutY have an elevated frequency of CG-to-AT transversion mutations (18); these are reduced by excess MutS, suggesting that 8-oxoG/A mismatches are also subject to MMR (23). As shown in Fig. ​Fig.3,3, the interactions between Vsr and MutL and between MutH and MutL increase in a mutY cell (stippled bars). Other interactions, such as MutS dimerization, are unaffected (not shown).Open in a separate windowFIG. 3.Effects of mutY and mutT deletions on protein-protein interactions in the bacterial two-hybrid assay. Results are in units of β-galactosidase, relative to the level in the wild type, in mutT (solid) and mutY (stippled) derivatives of BTH101 cotransformed with pT18 and pT25 vectors, pT18-mutH and pT25-mutL, pT18-vsr and pT25-mutL, or pT18-mutS and pT25-mutS (n = 3).8-OxoG/A mismatches also arise by incorporation of oxidized dGTP opposite A during DNA replication. The MutT nuclease minimizes this by removing oxidized dGTP from the nucleotide pool. The high frequency of AT-to-CG mutations in mutT strains is unaffected by the status of the MMR system (7, 21, 23), possibly because these 8-oxoG/A mispairs are in a conformation that MutS does not recognize. As shown in Fig. ​Fig.3,3, neither the interaction between MutL and Vsr nor that between MutL and MutH is elevated in a mutT strain (solid bars).These data show that mismatches which attract MutS and MutL increase the interaction of MutL with MutH in vivo. Although these mismatches are not subject to VSP repair, they also increase the interaction between MutL and Vsr. The simplest interpretation is that a MutS-MutL complex recruits MutH and Vsr to the DNA independent of the identity of the mismatch. MutS and MutL could then clear the mismatch, delivering the (activated) endonuclease to its specific target site, no matter how far away it is.Interaction of MutL with MutH, leading to MMR, is probably the default option. However, the MutS-MutL complex may recruit other repair proteins, such as Vsr or UvrB (20), to lesions that are poorly processed by MMR. The T/G mismatch in hemimethylated CTWGG sequences may be one such site. Vsr is expressed at very low levels in growing cells (14), so this recruitment would enhance VSP repair. However, recruitment of Vsr to other lesions would reduce VSP repair. For example, recruitment of Vsr by MutL to 2AP/C lesions (Fig. ​(Fig.2)2) could explain why CCWGG sites are hotspots for 2AP-induced mutations (4, 19).We have argued that Vsr is kept at low levels while DNA is replicating to avoid interference with MMR (14). However, if, as we suggest here, MutS and MutL are needed to recruit scarce Vsr to its target sequence, this argument loses its merit. It seems more likely that Vsr levels are kept low to avoid CTWGG-to-CCWGG mutations; Vsr creates these mutations by converting T/G mismatches formed at CTAGG sites by errors in DNA replication to CG (3, 6, 16). Vsr levels rise in nongrowing cells (14), when mutagenesis is no longer a risk. Under these circumstances, it is likely that MutS and MutL are no longer required for efficient VSP repair.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

The ACTH/MSH (4-9) analog Org2766 improves retrieval of information after a fimbria fornix transection

The fimbria fornix of male Wistar rats was transected unilaterally after they had been successfully trained in the Morris maze and the passive avoidance task. Sham-operated and lesioned animals were treated either with Org2766 or saline for two weeks. Subsequently, the performance of all groups was tested again starting two days after the last treatment. The lesioned animals showed a deficit in performance in both tasks, indicating interference of the lesion with retrieval of information. Org2766 improved the poor performance of the lesioned animals in the Morris maze, but not in the passive avoidance task.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.