Rescue of arrested replication forks by homologous recombination

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5′-3′ and the other with 3′-5′ polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⋅T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a “synaptic pathway” involving a three-stranded nucleoprotein intermediate, rather than by a “helicase pathway” involving the separation and reannealing of DNA strands.

Force and kinetic barriers to unzipping of the DNA double helix

A theory of the unzipping of double-stranded DNA is presented and is compared to recent micromanipulation experiments. It is shown that the interactions that stabilize the double helix and the elastic rigidity of single strands simply determine the sequence-dependent ≈12-pN force threshold for DNA strand separation. Using a semimicroscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.

Computer-modeling origin of a simple genetic apparatus

This computer simulation is based on a model of the origin of life proposed by H. Kuhn and J. Waser, where the evolution of short molecular strands is assumed to take place in a distinct spatiotemporal structured environment. In their model, the prebiotic situation is strongly simplified to grasp essential features of the evolution of the genetic apparatus without attempts to trace the historic path. With the tool of computer implementation confining to principle aspects and focused on critical features of the model, a deeper understanding of the model's premises is achieved. Each generation consists of three steps: (i) construction of devices (entities exposed to selection) presently available; (ii) selection; and (iii) multiplication of the isolated strands (R oligomers) by complementary copying with occasional variation by copying mismatch. In the beginning, the devices are single strands with random sequences; later, increasingly complex aggregates of strands form devices such as a hairpin-assembler device which develop in favorable cases. A monomers interlink by binding to the hairpin-assembler device, and a translation machinery, called the hairpin-assembler-enzyme device, emerges, which translates the sequence of R1 and R2 monomers in the assembler strand to the sequence of A1 and A2 monomers in the A oligomer, working as an enzyme.

A single G-to-C change causes human centromere TGGAA repeats to fold back into hairpins.

Recently, we established that satellite III (TGGAA)n tandem repeats, which occur at the centromeres of human chromosomes, pair with themselves to form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. In separate studies, we have also established that the GCA triplet does not form bimolecular (GCA)2 motifs but instead promotes the formation of hairpins containing a GCA-turn motif in which the loop contains a single cytidine closed by a sheared G.A pair. Since TGCAA is the most frequent variant of TGGAA found in satellite III repeats, we reasoned that the potential of this variant to form GCA-turn miniloop fold-back structures might be an important factor in modulating the local structure in natural (TGGAA)n repeats. We report here the NMR-derived solution structure of the heptadecadeoxynucleotide (G)TGGAATGCAATGGAA(C) in which a central TGCAA pentamer is flanked by two TGGAA pentamers. This 17-mer forms a rather unusual and very stable hairpin structure containing eight base pairs in the stem, only four of which are Watson-Crick pairs, and a loop consisting of a single cytidine residue. The stem contains a (GGA)2 motif with intercalative 14G/4G stacking between two sheared G.A base pairs; the loop end of the stem consists of a sheared 8G.10A closing pair with the cytosine base of the 9C loop stacked on 8G. The remarkable stability of this unusual hairpin structure (Tm = 63 degrees C) suggests that it probably plays an important role in modulating the folding of satellite III (TGGAA)n repeats at the centromere.

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

Initiation sites of protein folding by NMR analysis.

Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable.

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.

Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.

Activating transcription from single stranded DNA.

Sequence specific regulators of eukaryotic gene expression, axiomatically, act through double stranded DNA targets. Proteins that recognize DNA cis-elements as single strands but for which compelling evidence has been lacking to indicate in vivo involvement in transcription are orphaned in this scheme. We sought to determine whether sequence specific single strand binding proteins can find their cognate elements and modify transcription in vivo by studying heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the single stranded sequence (CCCTCCCCA; CT-element) of the human c-myc gene in vitro. To monitor its DNA binding in vivo, the ability of hnRNP K to activate a reporter gene was amplified by fusion with the VP16 transactivation domain. This chimeric protein was found to transactivate circular but not linear CT-element driven reporters, suggesting that hnRNP K recognizes a single strand region generated by negative supercoiling in circular plasmid. When CT-elements were engineered to overlap with lexA operators, addition of lexA protein, either in vivo or in vitro, abrogated hnRNP K binding most likely by preventing single strand formation. These results not only reveal hnRNP K to be a single strand DNA binding protein in vivo, but demonstrate how a segment of DNA may modify the transcriptional activity of an adjacent gene through the interconversion of duplex and single strands.

Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.