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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.

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Related in: MedlinePlus

Characterization of the SeqAΔ(41–59)-A25R mutant. (a) Elution profiles of SeqA-A25R (20 315 Da) and SeqAΔ(41–59)-A25R (18 513 Da) over a Superdex-75 size exclusion chromatography column (GE Healthcare). Elution volumes of albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) are indicated. (b) Electrophoretic mobility shift assay of SeqAΔ(41–59)-A25R with DNAs containing two hemimethylated GATC sequences separated by a variable number of base pairs (X). The left-most lane contains an equimolar mixture of DNAs with 5, 7, 12, 21, 25 and 34 base pairs between the two GATC sites but no protein. (c) From left to right, flow cytometry profiles of a wild-type strain, the ΔseqA::tet strain and the ΔseqA::tet strain transformed with pET11a plasmids encoding wild-type SeqA, SeqAΔ(41–59), and SeqAΔ(41–59)-A25R. Wild-type SeqA and SeqAΔ(41–59) restore replication synchrony, however synchrony is lost upon protein over-expression. Conversely, SeqAΔ(41–59)-A25R only restores replication synchrony upon protein overexpression by addition of 25 μM IPTG.
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Figure 1: Characterization of the SeqAΔ(41–59)-A25R mutant. (a) Elution profiles of SeqA-A25R (20 315 Da) and SeqAΔ(41–59)-A25R (18 513 Da) over a Superdex-75 size exclusion chromatography column (GE Healthcare). Elution volumes of albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) are indicated. (b) Electrophoretic mobility shift assay of SeqAΔ(41–59)-A25R with DNAs containing two hemimethylated GATC sequences separated by a variable number of base pairs (X). The left-most lane contains an equimolar mixture of DNAs with 5, 7, 12, 21, 25 and 34 base pairs between the two GATC sites but no protein. (c) From left to right, flow cytometry profiles of a wild-type strain, the ΔseqA::tet strain and the ΔseqA::tet strain transformed with pET11a plasmids encoding wild-type SeqA, SeqAΔ(41–59), and SeqAΔ(41–59)-A25R. Wild-type SeqA and SeqAΔ(41–59) restore replication synchrony, however synchrony is lost upon protein over-expression. Conversely, SeqAΔ(41–59)-A25R only restores replication synchrony upon protein overexpression by addition of 25 μM IPTG.

Mentions: Host strain BL21DE3/pLysS was made ΔseqA::tet by P1 transduction with lysate from MM294 ΔseqA::tet (a kind gift from Dr Kleckner). This strain was supplemented with pET11a derivatives encoding: SeqA (pSS1), SeqA-A25R (pAG8015), SeqAΔ(41–59) (pAG8023), SeqAΔ(41–59)-A25R (pAG8033), SeqA-R70S-R73S (pAG8270), SeqA-A25R-R70S-R73S (pAG8268) and SeqAΔ(41–59)-A25R-R70S-R73S (pAG8269). In each case, the average number of origins per cell was determined by the flow cytometry ‘run-off’ method with modifications (33). Overnight cultures were grown in the absence or presence of 25 μM IPTG at 37°C in M63 minimal media with the appropriate antibiotics. The overnight cultures were diluted to an OD600 of 0.02 and grown to an OD600 ∼0.1 prior to incubation for 3 h with rifampicin (200 μg/ml) and cephalexin (36 μg/ml). After fixing with 77% ethanol, cells were analyzed in a Bryte SH flow cytometer (Biorad) using WinBryte software (Figure 1) and in an Apogee A40-Mini FCM flow cytometer (Apogee Flow Systems) using Apogee Histogram Software version 1.94 (Figure 4D).Figure 1.


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)

Characterization of the SeqAΔ(41–59)-A25R mutant. (a) Elution profiles of SeqA-A25R (20 315 Da) and SeqAΔ(41–59)-A25R (18 513 Da) over a Superdex-75 size exclusion chromatography column (GE Healthcare). Elution volumes of albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) are indicated. (b) Electrophoretic mobility shift assay of SeqAΔ(41–59)-A25R with DNAs containing two hemimethylated GATC sequences separated by a variable number of base pairs (X). The left-most lane contains an equimolar mixture of DNAs with 5, 7, 12, 21, 25 and 34 base pairs between the two GATC sites but no protein. (c) From left to right, flow cytometry profiles of a wild-type strain, the ΔseqA::tet strain and the ΔseqA::tet strain transformed with pET11a plasmids encoding wild-type SeqA, SeqAΔ(41–59), and SeqAΔ(41–59)-A25R. Wild-type SeqA and SeqAΔ(41–59) restore replication synchrony, however synchrony is lost upon protein over-expression. Conversely, SeqAΔ(41–59)-A25R only restores replication synchrony upon protein overexpression by addition of 25 μM IPTG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Characterization of the SeqAΔ(41–59)-A25R mutant. (a) Elution profiles of SeqA-A25R (20 315 Da) and SeqAΔ(41–59)-A25R (18 513 Da) over a Superdex-75 size exclusion chromatography column (GE Healthcare). Elution volumes of albumin (67 kDa), ovoalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa) are indicated. (b) Electrophoretic mobility shift assay of SeqAΔ(41–59)-A25R with DNAs containing two hemimethylated GATC sequences separated by a variable number of base pairs (X). The left-most lane contains an equimolar mixture of DNAs with 5, 7, 12, 21, 25 and 34 base pairs between the two GATC sites but no protein. (c) From left to right, flow cytometry profiles of a wild-type strain, the ΔseqA::tet strain and the ΔseqA::tet strain transformed with pET11a plasmids encoding wild-type SeqA, SeqAΔ(41–59), and SeqAΔ(41–59)-A25R. Wild-type SeqA and SeqAΔ(41–59) restore replication synchrony, however synchrony is lost upon protein over-expression. Conversely, SeqAΔ(41–59)-A25R only restores replication synchrony upon protein overexpression by addition of 25 μM IPTG.
Mentions: Host strain BL21DE3/pLysS was made ΔseqA::tet by P1 transduction with lysate from MM294 ΔseqA::tet (a kind gift from Dr Kleckner). This strain was supplemented with pET11a derivatives encoding: SeqA (pSS1), SeqA-A25R (pAG8015), SeqAΔ(41–59) (pAG8023), SeqAΔ(41–59)-A25R (pAG8033), SeqA-R70S-R73S (pAG8270), SeqA-A25R-R70S-R73S (pAG8268) and SeqAΔ(41–59)-A25R-R70S-R73S (pAG8269). In each case, the average number of origins per cell was determined by the flow cytometry ‘run-off’ method with modifications (33). Overnight cultures were grown in the absence or presence of 25 μM IPTG at 37°C in M63 minimal media with the appropriate antibiotics. The overnight cultures were diluted to an OD600 of 0.02 and grown to an OD600 ∼0.1 prior to incubation for 3 h with rifampicin (200 μg/ml) and cephalexin (36 μg/ml). After fixing with 77% ethanol, cells were analyzed in a Bryte SH flow cytometer (Biorad) using WinBryte software (Figure 1) and in an Apogee A40-Mini FCM flow cytometer (Apogee Flow Systems) using Apogee Histogram Software version 1.94 (Figure 4D).Figure 1.

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