Clamp loaders

B Guenther, R Onrust, A Sail, M O Donnell, J Kuriyan. Crystal structure of the 5 subunit of the clamp-loader complex of E. coli DNA polymerase III. Cell 91 335-345, 1997. 

Two other protein complexes also function in eukaryotic DNA replication. RPA (replication protein A) is a eukaryotic single-stranded DNA-binding protein, equivalent in function to the E. coli SSB protein. RFC (replication/actor (J) is a clamp loader for PCNA and facilitates the assembly of active replication complexes. The subunits of the RFC complex have significant sequence similarity to the subunits of the bacterial clamploading (y) complex. 

Jeruzalmi, D., O DonneB, M., Kuriyan, J. (2002) Clamp loaders and sliding clamps. Curr. Opin. Struct. Biol. 12, 217-224. Summary of some of the elegant work elucidating how clamp loaders function. 

High-fidelity chromosomal replication in E. coli is executed by a multicomponent complex referred to as DNA polymerase III holoenzyme (see Fig. 4a) (18-21). Pol 111 holoenzyme consists of three main subcomponents Pol 111 core, 3-clamp, and y-complex clamp-loader. Pol 111 core is the replicative DNA polymerase that consists of three subunits (a, e, 0) a exhibits DNA polymerase activity, e performs 3 -5 exonuclease activity necessary for proofreading, and the function of 0 is currently unclear. 

The y-complex clamp-loader is a heteropentameric ATPase responsible for assembling p onto DNA at primed sites (26-28). The clamp loading process is illustrated in Fig. 5b. The y-complex binds and opens the ring-shaped P-clamp in the presence of ATP. The ATP-bound y-complex selectively binds to a primed site that stimulates ATP hydrolysis and results in release of the clamp-loader and closure of P around DNA (20). 

DnaB is the replicative helicase that unwinds the parental duplex DNA ahead of the replication fork (see Fig. 4a) (40). DnaB encircles the lagging strand as a homohexamer and uses the energy of ATP hydrolysis to unwind DNA with 5 -3 polarity see also DNA Helicases, Chemistry and Mechanisms of). DnaB is connected to Pol III holoenzyme via its interaction with the X subunit of the clamp loader (41). This interaction greatly stimulates DnaB activity at the replication fork (42). DnaB also binds to and stimulates primase, which is a specialized RNA polymerase that synthesizes RNA primers approximately 12 nucleotides in length to initiate DNA synthesis (1, 43). 

Clamp-loader (heteropentamer) 297.1 Assembles sliding-clamps, protein trafficking... 

Bowman GD, Goedken ER, Kazmirski SL, O Donnell M, Kuriyan J. DNA polymerase clamp loaders and DNA recognition. FEBS Lett. 2005 579 863-867. 

Bowman GD, O Donnell M, Kuriyan J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 2004 429 724-730. 

Neuwald AF. Hypothesis bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine. Nucleic Acids Res. 2006 34 5280-5290. 

O Donnell M, Jeruzalmi D, Kuriyan J. Clamp loader structure predicts the architecture of DNA polymerase III holoenzyme and RFC. Curr. Biol. 2001 11 935-946. 

Bermudez VP, Lindsey-Boltz LA, Cesare AJ, Maniwa Y, Griffith JD, Hurwitz J, Sancar A. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRadl7-replication factor C complex in vitro. Proc. Natl. Acad. Sci. U.S.A. 2003 100(4) 1633-1638. 

Ellison V, Stillman B. Biochemical characterization of DNA damage checkpoint complexes clamp loader and clamp complexes with specificity for 5 recessed DNA. PLoS. Biol. 2003 1(2) E33. Kumagai A, Lee J, Yoo HY, Dunphy WG. TopBPl activates the ATR-ATRIP complex. Cell 2006 124(5) 943-955. 

Replication requires coordination between helicase, primase, SSBPs, two polymerases, clamps, loaders, topoisomerases, ligases and RNAses. 

Figure 28.28 DNA polymerase holoenzyme. Each holoenzyme consists of two copies of the polymerase core enzyme, which comprises the n. e, and 0 subunits and two copies of the (i subunit, linked to a central structure. The central structure includes the clamp loader complex and the hexameric helicase DnaB.

Repair synthesis is executed by components of the replication machinery-the clamp loader RFC, the processivity factor PCNA, and the replicative Pols 8/e [51, 94], Recent studies have additionally implicated the translesion synthesis Pol k in repair synthesis [95], but what role the different polymerases play in the process remains to be elucidated. Following the fill-in reaction, the nick in the DNA is sealed by XRCC1-DNA ligase 3a in all stages of the cell cyde and by DNA ligase 1 in proliferating cells [96], thus completing the NER process. 

Clamps and clamp loaders - Protein from the DNA polymerase III holoenzyme... 

X, P,y - This group of proteins is called the J complex (also called the clamp loader) (Figure 24.21). It is composed of one copy of all the proteins except of which there are 2 or 3 copies. The clamp loader wraps the clamp onto the DNA. Figure 24.21 shows some of the steps in this process. 

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