Search PubMed⌕ Search

Biomedical subjects

Susan T Lovett

Publications and source records attributed to Susan T Lovett.

15 recordsLinked to original sources
PubMed2006

RecA-independent recombination is efficient but limited by exonucleases.

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in Escherichia coli is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI(-) RecJ(-) strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coli encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.

Crossing Over, Genetic↗
PubMed2006

Replication arrest-stimulated recombination: Dependence on the RecA paralog, RadA/Sms and translesion polymerase, DinB.

Difficulties in replication can lead to breakage of the fork. Recombinational reactions restore the integrity of the fork through strand-invasion of the broken chromosome with its sister. If this occurs in the context of repeated DNA sequences, genetic rearrangements can result. We have proposed that this process accounts for stimulation of chromosomal rearrangements by mutations in Escherichia coli's replicative DNA helicase, DnaB. At its permissive temperature for growth, a dnaB107 mutant is a 1000-fold more likely to experience a deletion of a 787bp tandem repeated segment inserted in the E. coli chromosome than is a wild-type strain. We have previously shown that enhanced deletion in a dnaB107 strain is reduced in recA, recB and recG102 (formerly known as radC102) derivatives. Here I show that this enhanced recombination is dependent on other factors: the RuvA Holliday junction helicase, the RecJ single-strand DNA exonuclease, the RadA/Sms RecA-paralog protein of unknown function and, surprisingly, the DinB translesion polymerase. The requirement for these factors in DnaB-stimulated rearrangements is much greater than that observed for recombinational events such as P1 transduction. This may be because strand invasion into the repeats limits the extent of heteroduplex DNA that can be formed in the initial stage of recombination. I propose that RadA, RecG and RuvAB are critically required to stabilize the strand-invasion intermediate and that DinB polymerase extends the invading 3' strand to aid in re-initiation. The role of DinB in bacteria may be analogous to translesion DNA polymerase eta in eukaryotes, recently shown to aid recombination.

DNA Polymerase II↗
PubMed2006

DNA repeat rearrangements mediated by DnaK-dependent replication fork repair.

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.

DNA Polymerase III↗
PubMed2006

RecJ exonuclease: substrates, products and interaction with SSB.

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5'-3' direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5' single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5' tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading approximately 1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5' phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.

DNA↗
PubMed2006

The role of replication initiation control in promoting survival of replication fork damage.

Dam methylase mutants were recovered in a screen for mutants sensitive to UV irradiation or mild inhibition of replication elongation. Dam's role in tolerance of DNA damage is to provide binding sites for SeqA, because seqA mutants showed similar sensitivity that was genetically epistatic to dam. The sensitivity of seqA mutants to UV irradiation and to the replication inhibitors hydroxyurea (HU) and azidothymidine (AZT) was suppressed by alleles of dnaA that reduce the efficiency of replication initiation. These results suggest that for survival of replication fork damage, SeqA's repression of replication initiation is more important than its effects on nucleoid organization. Convergence of forks upon DNA damage is a likely explanation for seqA mutant sensitivity, because its poor survival of UV was suppressed by reducing secondary initiation through minimal medium growth. Surprisingly, growth in minimal medium reduced the ability of seqA+ strains to form colonies in the presence of low levels of AZT. Double dnaA seqA mutants exhibited plating efficiencies much superior to wild-type strains during chronic low-level AZT exposure in minimal medium. This suggests that mild inhibition of replication fork progression may actively restrain initiation such that seqA+ strains fail to recover initiation capacity after sustained conditions of replication arrest.

Bacterial Outer Membrane Proteins↗
PubMed2005

Cis and trans-acting effects on a mutational hotspot involving a replication template switch.

A natural mutational hotspot in the thyA gene of Escherichia coli accounts for over half of the mutations that inactivate this gene, which can be selected by resistance to the antibiotic trimethoprim. This T to A transversion, at base 131 of the coding sequence, occurs within a 17 bp quasi-palindromic sequence. To clarify the mechanism of mutagenesis, we examine here cis and trans-acting factors affecting thyA131 mutational hotspot activity at its natural location on the E.coli chromosome. Confirming a template-switch mechanism for mutagenesis, an alteration that strengthens base-pairing between the inverted repeat DNA sequences surrounding the hotspot stimulated mutagenesis and, conversely, mutations that weakened pairing reduced hotspot activity. In addition, consistent with the idea that the hotspot mutation is templated from DNA synthesis from mispaired strands of the inverted repeats, co-mutation of multiple sites within the quasipalindrome was observed as predicted from the DNA sequence of the corresponding repeat. Surprisingly, inversion of the thyA operon on the chromosome did not abolish thyA131 hotspot mutagenesis, indicating that mutagenesis at this site occurs during both leading and lagging-strand synthesis. Loss of the SOS-induced DNA polymerases PolII, PolIV, and PolV, caused a marked increase in the hotspot mutation rate, indicating a heretofore unknown and redundant antimutagenic effect of these repair polymerases. Hotspot mutagenesis did not require the PriA replication restart factor and hence must not require fork reassembly after the template-switch reaction. Deficiency in the two major 3' single-strand DNA exonucleases, ExoI and ExoVII, stimulated hotspot mutagenesis 30-fold and extended the mutagenic tract, indicating that these exonucleases normally abort a large fraction of premutagenic events. The high frequency of quasipalindrome-associated mutations suggests that template-switching occurs readily during chromosomal replication.

Base Pairing↗
PubMed2005

Filling the gaps in replication restart pathways.

Recent work by clarifies two mechanisms of replication fork assembly in E. coli: in vitro, the presence of a leading nascent strand dictates a PriA/B DnaT-dependent mechanism, whereas a gapped fork specifies PriC-mediated restart.

Adenosine Triphosphatases↗
PubMed2005

A bacterial G protein-mediated response to replication arrest.

To define factors in E. coli promoting survival to replication fork stress, we isolated insertion mutants sensitive to replication inhibitors. One insertion caused partial loss of the universally conserved GTPase, obgE/yhbZ gene. Although obgE is essential for growth, our insertion allele supported viability until challenged with various replication inhibitors. A mutation designed to negate the GTPase activity of the protein produced similar phenotypes, but was genetically dominant. Synergistic genetic interactions with recA and recB suggested that chromosome breaks and regressed forks accumulate in obgE mutants. Mutants in obgE also exhibited asynchronous overreplication during normal growth, as revealed by flow cytometry. ObgE overexpression caused SeqA foci, normally localized to replication forks, to spread extensively within the cell. We propose that ObgE defines a pathway analogous to the replication checkpoint response of eukaryotes and acts in a complementary way to the RecA-dependent SOS response to promote bacterial cell survival to replication fork arrest.

Adenosine Triphosphatases↗
PubMed2004

Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences.

Mutations and rearrangements that occur by misalignment during DNA replication are frequent sources of genetic variation in bacteria. Dislocations between a replicating strand and its template at repetitive DNA sequences underlie the mechanism of these genetic events. Such misalignments can be transient or stable and can involve intramolecular or intermolecular DNA mispairing, even pairing across a replication fork. Paradoxically, these replication 'slippage' events both create and destroy repetitive sequences in bacterial genomes. This review catalogues several types of slippage errors, presents the cellular processes that act to limit them and discusses the consequences of this class of genetic events on the evolution of bacterial genomes and physiology.

Base Sequence↗
PubMed2003

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases.

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of approximately 100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.

Base Pair Mismatch↗
PubMed2003

Connecting replication and recombination.

Replication forks frequently break and must be repaired by recombination. A reconstituted reaction now allows the factors that coordinate conversion from a recombination intermediate back to a replication fork to be defined. The PriA protein plays a key role in this control.

DNA↗
PubMed2002

Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways.

We have developed an assay for intermolecular crossing over between circular plasmids carrying variable amounts of homology. Screens of Escherichia coli mutants demonstrated that known recombination functions can only partially account for the observed recombination. Recombination rates increased three to four orders of magnitude as homology rose from 25 to 411 bp. Loss of recA blocked most recombination; however, RecA-independent crossing over predominated at 25 bp and could be detected at all homology lengths. Products of recA-independent recombination were reciprocal in nature. This suggests that RecA-independent recombination may involve a true break-and-join mechanism, but the genetic basis for this mechanism remains unknown. RecA-dependent crossing over occurred primarily by the RecF pathway but considerable recombination occurred independent of both RecF and RecBCD. In many respects, the genetic dependence of RecA-dependent crossing over resembled that reported for single-strand gap repair. Surprisingly, ruvC mutants, in both recA(+) and recA mutant backgrounds, scored as hyperrecombinational. This may occur because RuvC preferentially resolves Holliday junction intermediates, critical to both RecA-dependent and RecA-independent mechanisms, to the noncrossover configuration. Levels of crossing over were increased by defects in DnaB helicase and by oxidative damage, showing that damaged DNA or stalled replication can initiate genetic recombination.

Crossing Over, Genetic↗
PubMed2002

Role for radA/sms in recombination intermediate processing in Escherichia coli.

RadA/Sms is a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and Lon protease. We examined mutations in the radA/sms gene of Escherichia coli for effects on conjugational recombination and sensitivity to DNA-damaging agents, including UV irradiation, methyl methanesulfonate (MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea (HU). Null mutants of radA were modestly sensitive to the DNA-methylating agent MMS and to the DNA strand breakage agent phleomycin, with conjugational recombination decreased two- to threefold. We combined a radA mutation with other mutations in recombination genes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC. A radA mutation was strongly synergistic with the recG Holliday junction helicase mutation, producing profound sensitivity to all DNA-damaging agents tested. Lesser synergy was noted between a mutation in radA and recJ, recQ, ruvA, ruvC, and recA for sensitivity to various genotoxins. For survival after peroxide and HU exposure, a radA mutation surprisingly suppressed the sensitivity of recA and recB mutants, suggesting that RadA may convert some forms of damage into lethal intermediates in the absence of these functions. Loss of radA enhanced the conjugational recombination deficiency conferred by mutations in Holliday junction-processing function genes, recG, ruvA, and ruvC. A radA recG ruv triple mutant had severe recombinational defects, to the low level exhibited by recA mutants. These results establish a role for RadA/Sms in recombination and recombinational repair, most likely involving the stabilization or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC.

ATP-Dependent Proteases↗