Binding of SeqA protein to hemi-methylated GATC sequences enhances their interaction and aggregation properties

The SeqA protein regulates chromosome initiation and is involved in segregation in Escherichia coli. One SeqA protein binds to two hemi-methylated GATC sequences to form a stable SeqA-DNA complex. We found that binding induced DNA bending, which was pronounced when the two sequences were on the same face of the DNA. Two SeqA molecules bound cooperatively to each pair of hemi-methylated sites when the spacing between the sites was < or = 30 bp. This cooperative binding was able to stabilize the binding of a wild type to a single hemi-methylated site, or mutant form of SeqA protein to hemi-methylated sites, although such binding did not occur without cooperative interaction. Two cooperatively bound SeqA molecules interacted with another SeqA bound up to 185 bp away from the two bound SeqA proteins, and this was followed by aggregation of free SeqA proteins onto the bound proteins. These results suggest that the stepwise interaction of SeqA proteins with hemi-methylated GATC sites enhances their interaction and leads to the formation of SeqA aggregates. Cooperative interaction followed by aggregation may be the driving force for formation of the SeqA foci that appear to be located behind replication forks.     

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.     

Positive supercoiling is generated in the presence of Escherichia coli SeqA protein

In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication. This protein is also suggested to have a role in chromosome organization. SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories. Loss of SeqA leads to increased negative supercoiling of the DNA. We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively. This means that binding of SeqA changes either the twist or the writhe of the DNA. The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo. The topology change performed by SeqA occurred also on unmethylated plasmids. It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy.     

The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC

The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro. The mechanism of inhibition is, however, not known. SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein. We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1. Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies. The affinity of SeqA for hemi-methylated oriC was higher than for fully methylated oriC. The binding was in both cases strongly cooperative. We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein-protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.     

Pre-replication assembly of E. coli replisome components

The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components alpha and tau, has been studied by immunofluorescence microscopy. The origin has been labelled through GFP-LacI bound near oriC. SeqA was located in the cell centre for one replication factory (RF) and at 1/4 and 3/4 positions in pre-divisional cells harbouring two RFs. The transition of central to 1/4 and 3/4 positions of SeqA appeared abrupt. Labelled thymidylate synthetase was found all over the cell, thus not supporting the notion of a dNTP-synthesizing complex exclusively localized near the RF. More DnaB, alpha and tau foci were found than expected. We have hypothesized that extra foci arise at pre-replication assembly sites, where the number of sites equals the number of origins, i.e. the number of future RFs. A reasonable agreement was found between predicted and found foci. In the case of multifork replication the number of foci appeared consistent with the assumption that three RFs are grouped into a higher-order structure. The RF is probably separate from the foci containing SeqA and the hemi-methylated SeqA binding sites because these foci did not coincide significantly with DnaB as marker of the RF. Co-labelling of DnaB and oriC revealed limited colocalization, indicating that DnaB did not yet become associated with oriC at a pre-replication assembly site. DnaB and tau co-labelled in the cell centre, though not at presumed pre-replication assembly sites. By contrast, alpha and tau co-labelled consistently suggesting that they are already associated before replication starts.     

Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences

The binding of SeqA protein to hemi-methylated GATC sequences (hemi-sites) regulates chromosome initiation and the segregation of replicated chromosome in Escherichia coli. We have used atomic force microscopy to examine the architecture of SeqA and the mode of binding of one molecule of SeqA to a pair of hemi-sites in aqueous solution. SeqA has a bipartite structure composed of a large and a small lobe. Upon binding of a SeqA molecule to a pair of hemi-sites, the larger lobe becomes visibly separated into two DNA binding domains, each of which binds to one hemi-site. The two DNA binding domains are held together by association between the two multimerization domains that make up the smaller lobe. The binding of each DNA binding domain to a hemi-site leads to bending of the bound DNA inwards toward the bound protein. In this way, SeqA adopts a dimeric configuration when bound to a pair of hemi-sites. Mutational analysis of the multimerization domain indicates that, in addition to multimerization of SeqA polypeptides, this domain contributes to the ability of SeqA to bind to a pair of hemi-sites and to its cooperative behavior.     

Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences

The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.     

Structural insights into the cooperative binding of SeqA to a tandem GATC repeat

SeqA is a negative regulator of DNA replication in Escherichia coli and related bacteria that functions by sequestering the origin of replication and facilitating its resetting after every initiation event. Inactivation of the seqA gene leads to unsynchronized rounds of replication, abnormal localization of nucleoids and increased negative superhelicity. Excess SeqA also disrupts replication synchrony and affects cell division. SeqA exerts its functions by binding clusters of transiently hemimethylated GATC sequences generated during replication. However, the molecular mechanisms that trigger formation and disassembly of such complex are unclear. We present here the crystal structure of a dimeric mutant of SeqA [SeqAΔ(41–59)-A25R] bound to tandem hemimethylated GATC sites. The structure delineates how SeqA forms a high-affinity complex with DNA and it suggests why SeqA only recognizes GATC sites at certain spacings. The SeqA–DNA complex also unveils additional protein–protein interaction surfaces that mediate the formation of higher ordered complexes upon binding to newly replicated DNA. Based on this data, we propose a model describing how SeqA interacts with newly replicated DNA within the origin of replication and at the replication forks.     

Anti-angiogenic peptides identified in thrombospondin type I domains

SeqA proteins of Escherichia coli bound to the hemimethylated GATC sequences (hemi-sites) interact with each other and eventually form an aggregate. SeqA foci, which are suggested to be formed by aggregation, play important roles in the regulation of chromosome replication and segregation. We found that aggregation of SeqA proteins was preceded by cooperative interactions between these proteins bound to hemi-sites. Positively charged amino acids in the hinge region, which connects the N-terminal and C-terminal domain of SeqA, were critical for SeqA aggregation on hemimethylated DNA. Although the substitution of positively charged amino acids with negatively charged or neutral amino acids maintained the binding and cooperative interaction of mutant proteins, these proteins were defective in aggregation and foci formation in vitro and in vivo, respectively. Our results suggest that in vivo SeqA foci were formed by aggregation following cooperative interactions between SeqA proteins bound to hemi-sites.     

A Mathematical Model for Timing the Release from Sequestration and the Resultant Brownian Migration of SeqA Clusters in <Emphasis Type="Italic">E. coli</Emphasis>

DNA replication in Escherichia coli is initiated by DnaA binding to oriC, the replication origin. During the process of assembly of the replication factory, the DnaA is released back into the cytoplasm, where it is competent to reinitiate replication. Premature reinitiation is prevented by binding SeqA to newly formed GATC sites near the replication origin. Resolution of the resulting SeqA cluster is one aspect of timing for reinitiation. A Markov model accounting for the competition between SeqA binding and methylation for one or several GATC sites relates the timing to reaction rates, and consequently to the concentrations of SeqA and methylase. A model is proposed for segregation, the motion of the two daughter DNAs into opposite poles of the cell before septation. This model assumes that the binding of SeqA and its subsequent clustering results in loops from both daughter nucleoids attached to the SeqA cluster at the GATC sites. As desequestration occurs, the cluster is divided in two, one associated with each daughter. As the loops of DNA uncoil, the two subclusters migrate apart due to the Brownian ratchet effect of the DNA loop.     

Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression.     

The initiator protein DnaA contributes to keeping new origins inactivated by promoting the presence of hemimethylated DNA

The Escherichia coli replication origin oriC and other regions with high numbers of GATC sites remain hemimethylated after replication much longer than regions with average numbers of GATC sites. The prolonged period of hemimethylation has been attributed to the presence of bound SeqA protein. Here, it was found that a GATC cluster inserted at the datA site, which binds large amounts of DnaA in vivo, did not become remethylated at all, unless the availability of the DnaA protein was severely reduced. Sequestration of oriC was also found to be affected by the availability of DnaA. The period of origin hemimethylation was reduced by approximately 30% upon a reduction in the availability of DnaA. The result shows that not only SeqA binding but also DnaA binding to newly replicated origins contributes to keeping them hemimethylated. It was also found that the number of SeqA foci increased in cells with a combination of DnaA-mediated protection and sequestration at the GATC::datA cluster.     

Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.     

The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology

The SeqA protein was identified as a factor that prevents reinitiation of newly replicated, hemimethylated origins. SeqA also seems to inhibit initiation of fully methylated origins, thus contributing to the regulation of chromosomal replication. The SeqA protein was found to bind to two sites in the left part of the origin, near the AT-rich region where strand separation takes place during initiation of replication. The same binding sites seemed to be preferred irrespective of whether the origin was in the newly replicated (hemimethylated) state or not. In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU. The restraint of supercoils by SeqA was, in contrast to that of HU, cooperative.     

Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.     

SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC

DnaA occupies only the three highest-affinity binding sites in E. coli oriC throughout most of the cell cycle. Immediately prior to initiation of chromosome replication, DnaA interacts with additional recognition sites, resulting in localized DNA-strand separation. These two DnaA-oriC complexes formed during the cell cycle are functionally and temporally analogous to yeast ORC and pre-RC. After initiation, SeqA binds to hemimethylated oriC, sequestering oriC while levels of active DnaA are reduced, preventing reinitiation. In this paper, we investigate how resetting of oriC to the ORC-like complex is coordinated with SeqA-mediated sequestration. We report that oriC resets to ORC during sequestration. This was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-affinity sites. Thus, during the sequestration period, SeqA repressed pre-RC assembly while ensuring resetting of E. coli ORC.     

SeqA protein aggregation is necessary for SeqA function.

The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.     

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication¹. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures². SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA³. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length⁴ and DNA ends (blunt or overhang)^5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqAΔ(41-59)-A25R) to obtain crystals suitable for structure determination.