Crossover Hotspot Instigator Research Articles

E. Coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

Distribution of RecBCD and AddAB recombination-associated genes among bacteria in 33 phyla

Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open questions about the distribution of recognized components of recombination machinery among bacteria and archaea. RecBCD helicase-nuclease plays a central role in recombination among Gammaproteobacteria like Escherichia coli; while bacteria in other phyla, like the Firmicute Bacillus subtilis, use the related AddAB complex. The activity of at least some of these complexes is controlled by short DNA sequences called crossover hotspot instigator (Chi) sites. When RecBCD or AddAB complexes encounter an autologous Chi site during unwinding, they introduce a nick such that ssDNA with a free end is available to invade another duplex. If homologous DNA is present, RecA-dependent homologous recombination is promoted; if not (or if no autologous Chi site is present) the RecBCD/AddAB complex eventually degrades the DNA. We examined the distribution of recBCD and addAB genes among bacteria, and sought ways to distinguish them unambiguously. We examined bacterial species among 33 phyla, finding some unexpected distribution patterns. RecBCD and addAB are less conserved than recA, with the orthologous recB and addA genes more conserved than the recC or addB genes. We were able to classify RecB vs. AddA and RecC vs. AddB in some bacteria where this had not previously been done. We used logo analysis to identify sequence segments that are conserved, but differ between the RecBC and AddAB proteins, to help future differentiation between members of these two families.

Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.

Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases.

Structural features of Chi recognition in AddAB with implications for RecBCD

AddAB and RecBCD-type helicase-nuclease complexes control the first stage of bacterial homologous recombination (HR) – the resection of double strand DNA breaks. A switch in the activities of the complexes to initiate repair by HR is regulated by a short, species-specific DNA sequence known as a Crossover Hotspot Instigator (Chi) site. It has been shown that, upon encountering Chi, AddAB and RecBCD pause translocation before resuming at a reduced rate. Recently, the structure of B.subtilis AddAB in complex with its regulatory Chi sequence revealed the nature of Chi binding and the paused translocation state. Here the structural features associated with Chi binding are described in greater detail and discussed in relation to the related E.coli RecBCD system.

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

Double-stranded DNA break repair by homologous recombination is initiated by resection of free DNA ends to produce a 3'-ssDNA overhang. In bacteria, this reaction is catalyzed by helicase-nuclease complexes such as AddAB in a manner regulated by specific recombination hotspot sequences called Crossover hotspot instigator (Chi). We have used magnetic tweezers to investigate the dynamics of AddAB translocation and hotspot scanning during double-stranded DNA break resection. AddAB was prone to stochastic pausing due to transient recognition of Chi-like sequences, unveiling an antagonistic relationship between DNA translocation and sequence-specific DNA recognition. Pauses at bona fide Chi sequences were longer, were nonexponentially distributed, and resulted in an altered velocity upon restart of translocation downstream of Chi. We propose a model for the recognition of Chi sequences to explain the origin of pausing during failed and successful hotspot recognition.

Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3' to 5' translocase) and RecD (5' to 3' translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5' to 3' rate is always faster than the 3' to 5' rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3' to 5' and 5' to 3' translocation, whereas RecD only regulates 5' to 3' translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding.

RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks

The RecBCD enzyme of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. It also degrades linear double-stranded DNA, protecting the bacteria from phages and extraneous chromosomal DNA. The RecBCD enzyme is, however, regulated by a cis-acting DNA sequence known as Chi (crossover hotspot instigator) that activates its recombination-promoting functions. Interaction with Chi causes an attenuation of the RecBCD enzyme's vigorous nuclease activity, switches the polarity of the attenuated nuclease activity to the 5' strand, changes the operation of its motor subunits, and instructs the enzyme to begin loading the RecA protein onto the resultant Chi-containing single-stranded DNA. This enzyme is a prototypical example of a molecular machine: the protein architecture incorporates several autonomous functional domains that interact with each other to produce a complex, sequence-regulated, DNA-processing machine. In this review, we discuss the biochemical mechanism of the RecBCD enzyme with particular emphasis on new developments relating to the enzyme's structure and DNA translocation mechanism.

Identification of DNA Motifs Implicated in Maintenance of Bacterial Core Genomes by Predictive Modeling

Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the “crossover hotspot instigator,” or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions.

Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.

Crossover hot-spot instigator (Chi) sequences in Escherichia coli occupy distinct recombination/transcription islands.

Crossover hot-spot instigator (Chi) sequences (5'-GCTGGTGG-3') are orientation-dependent, strand-specific sequences implicated in RecA-mediated DNA recombination. In Escherichia coli and Haemophilus influenzae Chi and Chi-like sequences preferentially locate to approx. 1kb recombination 'islands' in the mRNA-synonymous strands of open reading frames (ORFs). Since mRNA-synonymous strands follow Szybalski's transcription direction rule in being G-rich, and the average ORF is about 1kb, then, on this basis alone, Chi sequences are seen to reside in 1kb G-rich 'islands'. However, RecA preferentially binds GT-rich sequences, suggesting that genomic context might potentiate Chi action. Consistent with this, we report for E. coli that 1kb sequence windows with Chi near their centres are a distinct subset of total 1kb windows, the mRNA-synonymous strands being preferentially enriched in both G and T. Chi function might be particularly important for bacteria that survive high temperature and radiation. These often exist in habitats where recombination with E. coli DNA would be unlikely, so canonical Chi sequences might not confer a selective disadvantage in this respect. In general, Chi sequences are not more frequent in thermophilic bacteria and Deinococcus radiodurans, than in E. coli and other mesophilic bacteria. Only two of five thermophilic bacteria examined showed preferential location of Chi sequences to mRNA-synonymous strands. In the thermophile Methanococcus jannaschii, windows containing the canonical Chi sequence do not form a distinct subset. We suggest that in thermophilic bacteria and D. radiodurans the Chi function may be achieved by sequences that differ from the canonical Chi sequence, or that the number of these sequences is sufficient, or that the Chi function is unnecessary.

Correlation of chi orientation with transcription indicates a fundamental relationship between recombination and transcription.

Cross-over hot-spot instigator (Chi) sequences (5'-GCTGGTGG-3') are abundant, strand-specific, sequences, which locally increase recombination in Escherichia coli. Located within G-rich 'recombination islands', Chi orientations correlate with the orientations both of DNA replication and of transcription. Consistent with evidence from eukaryotic systems for a fundamental relationship between recombination and transcription, we find for E. coli Chi sequences, and for Haemophilus influenzae Chi-like sequences, that orientations correlate better with transcription than with replication. Complying with Szybalski's transcription direction rule, open reading frames in these prokaryotes have purine-rich mRNA-synonymous DNA strands. Hence, the G-richness of 'recombination islands' may reflect their correspondence with 'transcriptional islands' (genes). Comparison of a natural with the corresponding shuffled sequence, indicates a base order-dependent island unit of approx. 1kb. 1998 Elsevier Science B.V.