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.     

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.     

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.     

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.     

Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli

To demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.     

The mutH gene regulates the replication and methylation of the pMB1 origin.

DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.     

Insights into negative modulation of E. coli replication initiation from the structure of SeqA-hemimethylated DNA complex

The SeqA protein binds clusters of fully methylated or hemimethylated GATC sequences at oriC and negatively modulates the initiation of DNA replication. We find that SeqA can be proteolytically cleaved into an N-terminal multimerization and a C-terminal DNA-binding domain and have determined the crystal structure of the C-terminal domain in complex with a hemimethylated GATC site. SeqA makes direct hydrogen bonds and van der Waals contacts with the hemimethylated A-T base pair in addition to interactions with the surrounding bases and DNA backbone. The tetrameric protein-DNA complex found in the crystal suggests that SeqA binds multiple GATC sites on separate DNA duplexes, altering the overall DNA topology and sequestering oriC from replication initiation.     

Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation.

A mutant mini-P1 plasmid with increased copy number can be established in Dam- strains of Escherichia coli, where mini-P1 plasmid replication is normally blocked. Comparison of this plasmid and a plasmid driven by the host oriC replication origin showed that both origins are subject to control by methylation at two different levels. First, both origins appear to be subject to negative regulation acting at the level of hemimethylation. This probably involves the sequestration of the hemimethylated DNA produced by replication, as has been previously described for oriC. Second, both origins show a positive requirement for adenine methylation for efficient function in vivo. This conclusion is supported by the behavior of the P1 origin in an improved in vitro replication system. In vitro, where sequestration of hemimethylated DNA is not expected to occur, the hemimethylated P1 origin DNA was fully functional as a template. However, the activity of fully unmethylated DNA was severely restricted in comparison with that of either of the methylated forms. This in vitro uncoupling of the two effects of origin methylation suggests that two separate mechanisms are involved.     

Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division

Following initiation of chromosomal replication in Escherichia coli, newly initiated origins (oriCs) are prevented from further initiations by a mechanism termed sequestration. During the sequestration period (which lasts about one-third of a cell cycle), the origins remain hemimethylated. The SeqA protein binds hemimethylated oriC in vitro. In vivo, the absence of SeqA causes overinitiation and strongly reduces the duration of hemimethylation. The pattern of immunostained SeqA complexes in vivo suggests that SeqA has a role in organizing hemimethylated DNA at the replication forks. We have examined the effects of overexpressing SeqA under different cellular conditions. Our data demonstrate that excess SeqA significantly increases the time oriC is hemimethylated following initiation of replication. In some cells, sequestration continued for more than one generation and resulted in inhibition of primary initiation. SeqA overproduction also interfered with the segregation of sister nucleoids and caused a delay in cell division. These results suggest that SeqA's function in regulation of replication initiation is linked to chromosome segregation and possibly cell division.     

The Escherichia coli SeqA protein

The Escherichia coli SeqA protein contributes to regulation of chromosome replication by preventing re-initiation at newly replicated origins. SeqA protein binds to new DNA which is hemimethylated at the adenine of GATC sequences. Most of the cellular SeqA is found complexed with the new DNA at the replication forks. In vitro the SeqA protein binds as a dimer to two GATC sites and is capable of forming a helical fiber of dimers through interactions of the N-terminal domain. SeqA can also bind, with less affinity, to fully methylated origins and affect timing of “primary” initiations. In addition to its roles in replication, the SeqA protein may also act in chromosome organization and gene regulation.     

Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks.     

Lack of SeqA focus formation,specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2

In Escherichia coli wild-type cells newly formed origins cannot be reinitiated. The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA. Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene. Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo. The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA. The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork. In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC. The mutant protein was also incapable of restraining negative supercoils. Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.     

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.     

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.     

The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated

DNA from the E. coli replicative origin binds with high affinity to outer membrane preparations. Specific binding regions are contained within a 463 bp stretch of origin DNA between positions -46 and +417 on the oriC map. This region of DNA contains an unusually high number of GATC sites, the recognition sequence for the E. coli DNA adenine methylase. We show here that oriC DNA binds to membrane only when it is hemimethylated. The E. coli chromosomal origin is hemimethylated for 8-10 min after initiation of replication, and origin DNA binds to membranes only during this time period. Based on these results, we propose a speculative model for chromosome segregation in E. coli.     

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.     

Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re-initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA-less cells. The mutant origins underwent re-initiation, showing that the GATC sites in oriC are required for sequestration. Each re-initiation eventually gave rise to a cell containing an extra nucleoid. The three-nucleoid cells displayed one asymmetrically placed FtsZ-ring and divided into a two-nucleoid cell and a one-nucleoid cell. The three nucleoid-cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.     

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.     

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.