Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' → 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' → 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two- arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

Original languageEnglish (US)
Pages (from-to)10320-10326
Number of pages7
JournalBiochemistry
Volume36
Issue number33
DOIs
StatePublished - Aug 19 1997

Fingerprint

DnaB Helicases
Escherichia coli
Substrates
Adenosine Triphosphate
Fluorescence
Adenylyl Imidodiphosphate
Proteins
DNA Replication
Hydrolysis
Titration
Sedimentation
Stoichiometry
Free energy
Enzymes
Conformations
DNA
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork. / Jezewska, Maria J.; Rajendran, Surendran; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 36, No. 33, 19.08.1997, p. 10320-10326.

Research output: Contribution to journalArticle

@article{02191da643b447bc9a18ca257d00ce23,
title = "Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork",
abstract = "The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' → 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' → 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two- arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.",
author = "Jezewska, {Maria J.} and Surendran Rajendran and Wlodzimierz Bujalowski",
year = "1997",
month = "8",
day = "19",
doi = "10.1021/bi970712a",
language = "English (US)",
volume = "36",
pages = "10320--10326",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "33",

}

TY - JOUR

T1 - Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork

AU - Jezewska, Maria J.

AU - Rajendran, Surendran

AU - Bujalowski, Wlodzimierz

PY - 1997/8/19

Y1 - 1997/8/19

N2 - The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' → 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' → 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two- arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

AB - The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' → 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' → 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two- arm fork is not affected by the presence of the 3' arm. Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer. Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm. The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer. These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork. The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

UR - http://www.scopus.com/inward/record.url?scp=0030747747&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030747747&partnerID=8YFLogxK

U2 - 10.1021/bi970712a

DO - 10.1021/bi970712a

M3 - Article

VL - 36

SP - 10320

EP - 10326

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 33

ER -