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Biomedical subjects

D R Leach

Publications and source records attributed to D R Leach.

At least 19 recordsLinked to original sources
PubMed2001

Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans.

The recombination mechanisms that deal with double-strand breaks in organisms as diverse as phage, bacteria, yeast, and humans are remarkably conserved. We discuss conservation in the biochemical pathways required to recombine DNA ends and in the structure of the DNA products. In addition, we highlight that two fundamentally distinct broken DNA substrates exist and describe how they are repaired differently by recombination. Finally, we discuss the need to coordinate recombinational repair with cell division through DNA damage response pathways.

Animals↗
PubMed2001

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease.

DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.

2-Aminopurine↗
PubMed2000

The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli.

A 24 triplet TGG.CCA repeat array shows length- and orientation-dependent propagation when present in the plasmid pUC18. When TGG(24) is present as template for leading-strand synthesis, plasmid recovery is normal in all strains tested. However, when it acts as template for lagging-strand synthesis, plasmid propagation is seriously compromised. Plasmids carrying deletions in the 5' side of this sequence can be isolated and products carrying 15 TGG triplets do not significantly interfere with plasmid propagation. Mutations in sbcCD, mutS and recA significantly improve the recovery of plasmids with TGG(24) on the lagging-strand template. These findings suggest that TGG(24) can fold into a structure that can interfere with DNA replication in vivo but that TGG(15) cannot. Furthermore, since the presence of the MutS and SbcCD proteins are required for propagation interference, it is likely that stabilisation of mismatched base pairs and secondary structure cleavage are implicated. In contrast, there is no correlation of triplet repeat expansion and deletion instability with predicted DNA folding. These results argue for a dissociation of the factors affecting DNA fragility from those affecting trinucleotide repeat expansion-contraction instability.

Adenosine Triphosphatases↗
PubMed2000

Control of crossing over.

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.

Bacterial Proteins↗
PubMed2000

Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli.

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntington's disease and myotonic dystrophy. Here, we have investigated the effect of methyl-directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation-dependent repeat instability in wild-type E. coli and yeast, we observed no orientation dependence in MMR- E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild-type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR- cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS-dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR- cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.

Adenosine Triphosphatases↗
PubMed2000

Palindromes as substrates for multiple pathways of recombination in Escherichia coli.

A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication. Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination. We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome. The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization. This was done in both wt and sbcC backgrounds. Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways. Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome. In the absence of SbcCD, recombination still occurs, probably using a gap substrate. This process is also PriA independent, suggesting that there is no collapse of the replication fork. In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures.

Bacterial Proteins↗
PubMed1999

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli.

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.

Bacterial Proteins↗
PubMed1998

CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma.

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.

Abatacept↗
PubMed1998

The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA.

Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions. We have shown that the purified SbcCD protein of Escherichia coli cleaves a DNA hairpin. This cleavage does not require the presence of a free (3' or 5') DNA end and generates products with 3'-hydroxyl and 5'-phosphate termini. Electron microscopy of SbcCD has revealed the "head-rod-tail" structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member. This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication.

Bacterial Proteins↗
PubMed1998

Evidence for two preferred hairpin folding patterns in d(CGG).d(CCG) repeat tracts in vivo.

Unusual DNA secondary structures have been implicated in the expansion of trinucleotide repeat tracts that has been found to be responsible for a growing number of human inherited disorders and folate-sensitive fragile chromosome sites. By inserting trinucleotide repeat sequences into a palindromic clamp in lambda phage we are able to investigate their tendencies to form hairpins in vivo in any particular alignment and with odd or even numbers of repeat units in the hairpin. We previously showed that with d(CAG).d(CTG) repeat tracts there was a markedly greater tendency to form hairpins with even numbers of repeat units than with odd numbers, whereas d(GAC).d(GTC) repeats showed no such alternation despite having the same base composition. We expected that d(CGG).d(CCG) repeats, might show the same pattern as d(CAG).(CTG) repeats since they are also involved in trinucleotide repeat expansion disorders. The pattern was not so clear and we wondered whether this might be because d(CGG).d(CCG) repeats have more than one possible alignment in which they could self-anneal. We now present results for all three alignments, which suggest that while even-membered hairpins are preferred in the frame d(CGG).d(CCG), hairpins with odd numbers of trinucleotides are more stable in the frame d(GGC).d(GCC). In both cases the base-pair predicted to close the terminal loop of unpaired bases is 5'C.3'G which has previously been found to be a favoured loop-closing pair.

Cytosine↗
PubMed1998

Secondary structures in d(CGG) and d(CCG) repeat tracts.

Several studies have been made to elucidate the nature of secondary structures in the single strands of d(CGG).d(CCG) repeat tracts but with conflicting conclusions. Here, we review this work and attempt to come towards consensus. Some investigators find that the G-rich strand forms hairpins. Of these, some conclude that pairing is in the alignment d(GGC).d(GGC) with two Watson-Crick bonds and one G.G bond per duplex repeat, others conclude that the alignment is d(GCG).d(GCG) with two G.G bonds and one C.C bond per duplex repeat. Others find quadruplex formation and conclude that this is in the latter alignment with two G4-quartets per quadruplex repeat and C.C bonds. We investigate why these different results were obtained and conclude that quadruplexes are likely to form under physiological conditions. We argue that they are probably bonded in the alignment d(GGC).d(GGC) with one G4-quartet and two C.G.C.G. quartets per quadruplex repeat. The C-rich strand does not appear to form quadruplexes under physiological conditions but forms hairpins. Apparently, short hairpins adopt the alignment d(CCG).d(CCG) with mismatched cytosine residues stacked into the helix but with 15 or more repeat units, the dominant form is a distorted hairpin aligned as d(GCC).d(GCC) with unpaired cytosine residues possibly turned outwards and stacked in the minor groove.

Animals↗
PubMed1998

Replication strand preference for deletions associated with DNA palindromes.

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli. All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E. coli, in bacteriophage lambda and in high-copy-number pUC-based plasmids.

Bacteriophage lambda↗
PubMed1997

Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli.

The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.

Adenosine Triphosphate↗
PubMed1997

DIR: a novel DNA rearrangement associated with inverted repeats.

A novel DNA rearrangement has been characterised that is both a direct and inverted repeat. This rearrangement involves the 2-fold duplication of a plasmid sequence adjacent to the site of insertion of a long palindrome. The sequence of this rearrangement suggests that it has arisen by strand slippage from the leading to the lagging strand of the replication fork as a consequence of the presence of the long palindrome.

Deoxyribonuclease BamHI↗
PubMed1997

Different structures of selected and unselected araB-lacZ fusions.

Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system. Eighty-four independent araB-lacZ fusions were sequenced. All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC). Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers. The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions. Each araB-lacZ fusion contained two novel of DNA junctions. The MuR-lacZ junctions showed 'hot-spotting' according to established rules for Mu target selection. The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology. More extensive homology between MuR and araB sequences indicates potential STC isomerization a resolvable four-way structure analogous to a Holliday junction. These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biology process rather than a unitary biochemical reaction.

Bacteriophage mu↗
PubMed1997

Repair by recombination of DNA containing a palindromic sequence.

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild-type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild-type cells but not in sbcC mutants. Our results suggest that double-strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.

Bacterial Proteins↗
PubMed1996

Enhancement of antitumor immunity by CTLA-4 blockade.

One reason for the poor immunogenicity of many tumors may be that they cannot provide signals for CD28-mediated costimulation necessary to fully activate T cells. It has recently become apparent that CTLA-4, a second counterreceptor for the B7 family of costimulatory molecules, is a negative regulator of T cell activation. Here, in vivo administration of antibodies to CTLA-4 resulted in the rejection of tumors, including preestablished tumors. Furthermore, this rejection resulted in immunity to a secondary exposure to tumor cells. These results suggest that blockade of the inhibitory effects of CTLA-4 can allow for, and potentiate, effective immune responses against tumor cells.

Abatacept↗