Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases

Nina Yao, Jennifer Turner, Zvi Kelman, P. Todd Stukenberg, Frank Dean, David Shechter, Zhen Qiang Pan, Jerard Hurwitz, Mike O'Donnell

Research output: Contribution to journalArticle

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Abstract

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ('sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd ≈ 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 ≈ 24 min), β is more stable in solution (Kd < 60 pM) and on DNA (t1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd ≈ 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.

Original languageEnglish (US)
Pages (from-to)101-113
Number of pages13
JournalGenes to Cells
Volume1
Issue number1
StatePublished - 1996
Externally publishedYes

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Proliferating Cell Nuclear Antigen
DNA
Bacteriophage T4
DNA-Directed DNA Polymerase
Adenosine Triphosphate
replication initiator protein
Catalytic DNA
Escherichia coli
Proteins
Recycling
DNA Replication

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Yao, N., Turner, J., Kelman, Z., Todd Stukenberg, P., Dean, F., Shechter, D., ... O'Donnell, M. (1996). Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases. Genes to Cells, 1(1), 101-113.

Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases. / Yao, Nina; Turner, Jennifer; Kelman, Zvi; Todd Stukenberg, P.; Dean, Frank; Shechter, David; Pan, Zhen Qiang; Hurwitz, Jerard; O'Donnell, Mike.

In: Genes to Cells, Vol. 1, No. 1, 1996, p. 101-113.

Research output: Contribution to journalArticle

Yao, N, Turner, J, Kelman, Z, Todd Stukenberg, P, Dean, F, Shechter, D, Pan, ZQ, Hurwitz, J & O'Donnell, M 1996, 'Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases', Genes to Cells, vol. 1, no. 1, pp. 101-113.
Yao, Nina ; Turner, Jennifer ; Kelman, Zvi ; Todd Stukenberg, P. ; Dean, Frank ; Shechter, David ; Pan, Zhen Qiang ; Hurwitz, Jerard ; O'Donnell, Mike. / Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases. In: Genes to Cells. 1996 ; Vol. 1, No. 1. pp. 101-113.
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abstract = "Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ('sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd ≈ 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 ≈ 24 min), β is more stable in solution (Kd < 60 pM) and on DNA (t1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd ≈ 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.",
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T1 - Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases

AU - Yao, Nina

AU - Turner, Jennifer

AU - Kelman, Zvi

AU - Todd Stukenberg, P.

AU - Dean, Frank

AU - Shechter, David

AU - Pan, Zhen Qiang

AU - Hurwitz, Jerard

AU - O'Donnell, Mike

PY - 1996

Y1 - 1996

N2 - Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ('sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd ≈ 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 ≈ 24 min), β is more stable in solution (Kd < 60 pM) and on DNA (t1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd ≈ 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.

AB - Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ('sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. Results: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd ≈ 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 ≈ 24 min), β is more stable in solution (Kd < 60 pM) and on DNA (t1/2 ≈ 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd ≈ 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and β clamps on DNA necessitates an active process for their removal. The clamp loaders (RF-C and γ complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. Conclusions: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and β clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and β double as clamp unloaders presumably for the purpose of clamp recycling.

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