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

Chung YS, Brendler T, Austin S, Guarné A - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.

ABSTRACT
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 [SeqADelta(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.

Show MeSH

Related in: MedlinePlus

Modes of operation of SeqA at oriC and the replication forks. (a) Reduced spacing between GATC sites (shown as black dots) at oriC permits sequestration with only few SeqA molecules. Sequestration of the origin is abolished by lack of DNA binding (SeqA-N150A/N152A) and weakened by lack of SeqA oligomerization (SeqA-A25R, SeqA-T18E, SeqA-I21R). (b) SeqA works similarly at the replication forks. However, formation of a left handed SeqA-DNA filament at the forks compensates for the increased spacing between GATC sites. SeqA-DNA filaments are further stabilized by protein–protein interactions through their DNA-binding domains (marked with boxes on the bottom left panel). Mutations in this surface (SeqA-K66E/R70E) affect SeqA-induced DNA aggregation. Mutations destabilizing the dimerization domain (SeqA-D7K, SeqA-E9A) affect the functions of the SeqA dimer but not those of the SeqA filament and, hence, they likely have a more deleterious effect at the oriC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2691817&req=5

Figure 5: Modes of operation of SeqA at oriC and the replication forks. (a) Reduced spacing between GATC sites (shown as black dots) at oriC permits sequestration with only few SeqA molecules. Sequestration of the origin is abolished by lack of DNA binding (SeqA-N150A/N152A) and weakened by lack of SeqA oligomerization (SeqA-A25R, SeqA-T18E, SeqA-I21R). (b) SeqA works similarly at the replication forks. However, formation of a left handed SeqA-DNA filament at the forks compensates for the increased spacing between GATC sites. SeqA-DNA filaments are further stabilized by protein–protein interactions through their DNA-binding domains (marked with boxes on the bottom left panel). Mutations in this surface (SeqA-K66E/R70E) affect SeqA-induced DNA aggregation. Mutations destabilizing the dimerization domain (SeqA-D7K, SeqA-E9A) affect the functions of the SeqA dimer but not those of the SeqA filament and, hence, they likely have a more deleterious effect at the oriC.

Mentions: Conceivably, these weak interactions between αC helices from adjacent SeqA dimers may assist on compacting the SeqA-DNA filament (Figure 5). Interactions through αC may cross-link dimers, either bound to different DNA duplexes (intermolecular cross-link), or bound to the same DNA duplex (intramolecular cross-link). We favor the latter because previous studies of complexes with duplex containing multiple GATC sites do not show the presence of intermolecular cross-linked complexes (40).Figure 5.


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

Chung YS, Brendler T, Austin S, Guarné A - Nucleic Acids Res. (2009)

Modes of operation of SeqA at oriC and the replication forks. (a) Reduced spacing between GATC sites (shown as black dots) at oriC permits sequestration with only few SeqA molecules. Sequestration of the origin is abolished by lack of DNA binding (SeqA-N150A/N152A) and weakened by lack of SeqA oligomerization (SeqA-A25R, SeqA-T18E, SeqA-I21R). (b) SeqA works similarly at the replication forks. However, formation of a left handed SeqA-DNA filament at the forks compensates for the increased spacing between GATC sites. SeqA-DNA filaments are further stabilized by protein–protein interactions through their DNA-binding domains (marked with boxes on the bottom left panel). Mutations in this surface (SeqA-K66E/R70E) affect SeqA-induced DNA aggregation. Mutations destabilizing the dimerization domain (SeqA-D7K, SeqA-E9A) affect the functions of the SeqA dimer but not those of the SeqA filament and, hence, they likely have a more deleterious effect at the oriC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691817&req=5

Figure 5: Modes of operation of SeqA at oriC and the replication forks. (a) Reduced spacing between GATC sites (shown as black dots) at oriC permits sequestration with only few SeqA molecules. Sequestration of the origin is abolished by lack of DNA binding (SeqA-N150A/N152A) and weakened by lack of SeqA oligomerization (SeqA-A25R, SeqA-T18E, SeqA-I21R). (b) SeqA works similarly at the replication forks. However, formation of a left handed SeqA-DNA filament at the forks compensates for the increased spacing between GATC sites. SeqA-DNA filaments are further stabilized by protein–protein interactions through their DNA-binding domains (marked with boxes on the bottom left panel). Mutations in this surface (SeqA-K66E/R70E) affect SeqA-induced DNA aggregation. Mutations destabilizing the dimerization domain (SeqA-D7K, SeqA-E9A) affect the functions of the SeqA dimer but not those of the SeqA filament and, hence, they likely have a more deleterious effect at the oriC.
Mentions: Conceivably, these weak interactions between αC helices from adjacent SeqA dimers may assist on compacting the SeqA-DNA filament (Figure 5). Interactions through αC may cross-link dimers, either bound to different DNA duplexes (intermolecular cross-link), or bound to the same DNA duplex (intramolecular cross-link). We favor the latter because previous studies of complexes with duplex containing multiple GATC sites do not show the presence of intermolecular cross-linked complexes (40).Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.

ABSTRACT
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 [SeqADelta(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.

Show MeSH
Related in: MedlinePlus