Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.
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 beta 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 approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 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 approximately 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 beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma 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 beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.
Yao
N
N
Microbiology Department, Howard Hughes Medical Institute, Cornell University Medical College, New York, NY 10021, USA.
Turner
J
J
Kelman
Z
Z
Stukenberg
P T
PT
Dean
F
F
Shechter
D
D
Pan
Z Q
ZQ
Hurwitz
J
J
O'Donnell
M
M
D017122:Bacteriophage T4 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D012996:Solutions; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4; D004247:DNA; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase