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Chapter 11 : SeqA Protein Binding and the Replication Fork

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Abstract:

The membrane affinity of the SeqA protein tends to make it insoluble in whole-cell extracts unless the salt concentration is elevated. If SeqA protein binding forms a coherent filament of protein on the DNA with the sequences intervening between the GATC sites looped out, the newly replicated DNA would be organized and compacted as it emerges from the replication fork. The chromosome replication forks are not thought to move about the nucleoid as they progress around the chromosome. Chromosome segregation is a direct consequence of replication and occurs concomitantly with it. The DNA replication process itself drives DNA segregation, pushing the newly replicated DNA outward from the anchored replication forks toward opposite cell poles. The properties of the SeqA protein and its selective binding to the newly replicated DNA at the replication forks suggest that it might be directly involved in some or all of these processes. If the single-strand contact persisted through replication, newly replicated DNA would be uniquely marked by having protein subunits on one strand only. If this ‘‘hemidecorated’’ DNA were selfassociating, a mechanism similar to that proposed for SeqA might operate in the absence of any methylation signals.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Figures

Image of Figure 1.
Figure 1.

Replication creates hemimethylated GATC sequences. In , the sequence GATC is methylated on the adenine bases of both strands by the action of the Dam methylase. As the sequence is replicated, the newly synthesized strands are unmethylated, so that the product duplexes are hemimethylated for a period of time, until Dam methylase can act to restore full methylation.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 2.
Figure 2.

The effect of the spacing of GATC sites on SeqA binding. (Top) A series of oligonucleotide binding substrates have two hemimethylated GATC sequences in an otherwise randomly chosen sequence. The position of the first GATC sequence is fixed. The position of the second GATC sequence is varied to create a series of spacings between the adenine bases of 4 bp (when the GATCs are immediately adjacent to each other) to 34 bp. (Bottom) The results of electrophoretic mobility shift assays with purified SeqA protein. Binding is optimal when the two sites are very close (7 bp) or when they are on the same face of the helix (11, 21, and 31 bp).

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 3.
Figure 3.

The effects of SeqA binding. (A) A stretch of DNA duplex (single line) contains multiple, suitably spaced, hemimethylated GATC sequences (black boxes). It will bind SeqA efficiently. The bound form of the protein is thought, on theoretical grounds, to be a symmetrical dimer. Binding studies suggest that the product will be a SeqA filament with the intervening sequences looped out as shown below. (B) As the replication fork passes around the chromosome, fully methylated GATC sites (shaded boxes at left) become transiently hemimethylated (black boxes) until Dam methylase restores full methylation (shaded boxes at right). This would result in a tract of SeqA bound to the newly replicated DNA following the fork.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 4.
Figure 4.

Potential SeqA binding sites on the chromosome. The positions of 1,750 potential SeqA binding sites are shown that would bind SeqA if hemimethylated. The bars represent the positions of pairs of GATC sites which are less than 34 bp apart and have a spacing which is known to promote SeqA binding when hemimethylated (see Fig. 2 ). As somewhat larger spacings are likely to bind also, the actual number of potential binding sites is probably greater.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 5.
Figure 5.

A representation of the factory model for chromosome replication and segregation. (A) In slow-growing cells, the replication forks (black triangles) are anchored at the cell center in a replication “factory” which contains all the proteins necessary for ongoing replication. The old DNA (darkest coils) is fed into the factory, and the newly replicated DNA (lighter coils) emerges from it and is directed outward toward the cell poles, driven by the replication process itself. Condensation of the newly replicated DNA forms two new nucleoid structures (lighter coils). The newly replicated DNA is deposited on the inner faces of the nascent nucleoids, such that the origins (black disks) end up on the outer faces and the termini (black squares) on the inner faces of the completed nucleoids. (B) At fast growth rates, new rounds of replication initiate before the previous round is completed. New factory sites form in the cell at 1/4 and 3/4 positions, and the nucleoids segregate to daughter cells while still undergoing replication.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 6.
Figure 6.

The spatial distribution of replication forks in the cell. Cells growing at three different growth rates are illustrated. They correspond approximately to 80-, 40-, and 20-min generation times at 37°C. Above each cell, the configuration of the chromosome is diagrammed. The expected numbers and positions of the replication forks (forked lines) are shown for each cell. Newborn cells, late-cycle cells near division, and typical mid-cycle cells are illustrated. Note that at faster growth rates, some cells may have three pairs of forks, placed at the 1/4, center, and 3/4 cell positions as shown. However, if termination of replication occurs just after cell division, such cells will be rare, and most mid-cycle cells will have two pairs of forks at the 1/4 and 3/4 cell positions. At very fast growth rates, a third round of replication may start before cell division. In this case, most cells will have six pairs of forks, placed at the 1/8, 1/4, 3/8, 5/8, 3/4, and 7/8 positions.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 7.
Figure 7.

The numbers of SeqA foci per cell at three different growth rates. The numbers of SeqA foci in ∼100 cells were counted from cultures growing at three different growth rates at 30°C. Slow: cells growing in M63 glycerol medium; generation time 160 min. Moderate: cells growing in M63 glucose medium; generation time 50 min. Fast: cells growing in L broth; generation time 30 min. These generation times give cell cycle parameters roughly equivalent to those illustrated in Fig. 6 , although the actual times are greater here due to the low temperature which is required for GFP visualization.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 8.
Figure 8.

SeqA-GFP expressed in cells growing at three different growth rates at 30°C. (a) Cells growing in minimal glycerol medium (160-min doubling time). (b) Cells growing in minimal glucose medium (50-min doubling time). (c) Cells growing in LB broth (30-min doubling time).

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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Image of Figure 9.
Figure 9.

The replication factory and SeqA. The factory model assumes that the two replication forks for bidirectional replication are anchored at the cell center. The forks could be arranged in two different ways. (A) The forks are coordinated as shown. The old DNA feeds into the replication factory, and the newly synthesized DNA is channeled away in opposite directions along the long axis of the cell. As the newly synthesized DNA is hemimethylated, SeqA binds to it. Self-association of the bound SeqA keeps the two duplexes destined to form each nucleoid together as a coherent filament. This facilitates the movement of the products outward toward the cell poles. Further out toward the poles, full methylation of the DNA is restored, SeqA protein dissociates, and the DNA is condensed to form a new nucleoid structure. (B) An alternative configuration for the forks is shown. Here, it is assumed that the two polymerase dimers that are acting at the two forks isomerize so that each dimer synthesizes the leading strand of one fork and the lagging strand of the other ( ). This idea is attractive, because this configuration naturally holds the forks together, spatially coordinates the two duplexes that will form each nucleoid, and ensures that they emerge from the factory in opposite directions.

Citation: Brendler T, Austin S. 2005. SeqA Protein Binding and the Replication Fork, p 217-227. In Higgins N (ed), The Bacterial Chromosome. ASM Press, Washington, DC. doi: 10.1128/9781555817640.ch11
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