DNA clamp

A DNA clamp, also known as a sliding clamp, is a protein complex that serves as a processivity-promoting factor in DNA replication. As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of the rate-limiting steps in the DNA synthesis reaction is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase.^[2]

Top and side views of a homotrimer of the human PCNA sliding clamp (rainbow colored, N-terminus = blue, C-terminus = red) with double stranded DNA modeled through the central pore (magenta).^[1]

Cryo-EM structure of the DNA-bound PolD–PCNA processive complex

Structural basis for DNA binding by the PolD–PCNA complex

Structure

The DNA clamp is an α+β protein that assembles into a multimeric, six-domain ring structure that completely encircles the DNA double helix as the polymerase adds nucleotides to the growing strand.^[3] Each domain is in turn made of two β-α-β-β-β structural repeats.^[4] The DNA clamp assembles on the DNA at the replication fork and "slides" along the DNA with the advancing polymerase, aided by a layer of water molecules in the central pore of the clamp between the DNA and the protein surface. Because of the toroidal shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomers.

The DNA clamp fold is found in bacteria, archaea, eukaryotes and some viruses. In bacteria, the sliding clamp is a homodimer composed of two identical beta subunits of DNA polymerase III and hence is referred to as the beta clamp. In archaea^[5] and eukaryotes, it is a trimer composed of three molecules of PCNA. The T4 bacteriophage also uses a sliding clamp, called gp45 that is a trimer similar in structure to PCNA but lacks sequence homology to either PCNA or the bacterial beta clamp.^[3]

Taxon	Sliding clamp protein	Multimer state	Associated polymerase
Bacteria	beta subunit of pol III	dimer	DNA polymerase III
Archaea	archaeal PCNA	trimer	PolD
Eukaryote	PCNA	trimer	DNA polymerase delta
Caudovirales	IPR004190	trimer	Phage polymerase (e.g. T4)
Herpesviridae	non-clamp processivity factor	monomer	Virus-encoded polymerase

Bacterial

DNA polymerase III subunit beta
Crystallographic structure of the dimeric DNA polymerase beta subunit from E. coli.[6]
Identifiers
Organism	Escherichia coli
Symbol	dnaN
Entrez	948218
PDB	1MMI
RefSeq (Prot)	NP_418156
UniProt	P0A988
Other data
EC number	2.7.7.7
Chromosome	MG1655: 3.88 - 3.88 Mb
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The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. Two beta subunits are assembled around the DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-initiation complex. After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and epsilon subunits, which together create the complete holoenzyme.^[7][8][9] DNA polymerase III is the primary enzyme complex involved in prokaryotic DNA replication.

The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzes ATP to chaperone two beta subunits to bind to DNA. Once bound to DNA, the beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the αε polymerase complex. The α subunit possesses DNA polymerase activity and the ε subunit is a 3’-5’ exonuclease.^[9]

The beta chain of bacterial DNA polymerase III is composed of three topologically equivalent domains (N-terminal, central, and C-terminal). Two beta chain molecules are tightly associated to form a closed ring encircling duplex DNA.

DNA polymerase III, beta chain (whole protein) Identifiers Symbol DNA_polIII_beta InterPro IPR001001 SMART SM00480 SCOP2 2pol / SCOPe / SUPFAM Available protein structures: Pfam     PDB 1jqj​, 1jql​, 1mmi​, 1ok7​, 1unn​, 1vpk​, 2pol​, 3bep​, 3d1e​, 3d1f​, 3d1g​

DNA polymerase III, beta chain, N-terminal Identifiers Symbol DNA_pol3_beta Pfam PF00712 InterPro IPR022634 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

DNA polymerase III, beta chain, central Identifiers Symbol DNA_pol3_beta_2 Pfam PF02767 InterPro IPR022637 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

DNA polymerase III, beta chain, C-terminal Identifiers Symbol DNA_pol3_beta_3 Pfam PF02768 InterPro IPR022635 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

As a drug target

Certain NSAIDs (carprofen, bromfenac, and vedaprofen) exhibit some suppression of bacterial DNA replication by inhibiting bacterial DNA clamp.^[10]

Eukaryotic and archaeal

proliferating cell nuclear antigen
The assembled human DNA clamp, a trimer of the human protein PCNA.[11]
Identifiers
Symbol	PCNA
NCBI gene	5111
HGNC	8729
OMIM	176740
PDB	1axc
RefSeq	NM_002592
UniProt	P12004
Other data
EC number	2.7.7.7
Locus	Chr. 20 pter-p12
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The sliding clamp in eukaryotes is assembled from a specific subunit of DNA polymerase delta called the proliferating cell nuclear antigen (PCNA). The N-terminal and C-terminal domains of PCNA are topologically identical. Three PCNA molecules are tightly associated to form a closed ring encircling duplex DNA.

The sequence of PCNA is well conserved between plants, animals and fungi, indicating a strong selective pressure for structure conservation, and suggesting that this type of DNA replication mechanism is conserved throughout eukaryotes.^[12][13] In eukaryotes, a homologous, heterotrimeric "9-1-1 clamp" made up of RAD9-RAD1-HUS1 (911) is responsible for DNA damage checkpoint control.^[14] This 9-1-1 clamp mounts onto DNA in the opposite direction.^[15]

Archaea, probable evolutionary precursor of eukaryotes, also universally have at least one PCNA gene. This PCNA ring works with PolD, the single eukaryotic-like DNA polymerase in archaea responsible for multiple functions from replication to repair. Some unusual species have two or even three PCNA genes, forming heterotrimers or distinct specialized homotrimers.^[16] Archaeons also share with eukaryotes the PIP (PCNA-interacting protein) motif, but a wider variety of such proteins performing different functions are found.^[17]

PCNA is also appropriated by some viruses. The giant virus genus Chlorovirus, with PBCV-1 as a representative, carries in its genome two PCNA genes (Q84513, O41056) and a eukaryotic-type DNA polymerase.^[18] Members of Baculoviridae also encode a PCNA homolog (P11038).^[19]

Proliferating cell nuclear antigen, N-terminal domain Identifiers Symbol PCNA_N Pfam PF00705 InterPro IPR000730 PROSITE PDOC00265 SCOP2 1plq / SCOPe / SUPFAM Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB 1axc​C:1–125 1ge8​A:3–92 1isq​A:3–92 1iz4​A:3–92 1iz5​A:3–92 1plq​ :1–125 1plr​ :1–125 1rwz​A:1–114 1rxm​A:1–114 1rxz​A:1–114 1u76​C:1–125 1u7b​A:1–125 1ud9​C:11–100 1ul1​A:1–125 1vyj​G:1–125 1vym​C:1–125 1w60​B:1–125

Proliferating cell nuclear antigen, C-terminal domain Identifiers Symbol PCNA_C Pfam PF02747 InterPro IPR000730 PROSITE PDOC00265 SCOP2 1plq / SCOPe / SUPFAM Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB 1axc​C:127–254 1ge8​A:203–246 1isq​A:203–246 1iz4​A:203–246 1iz5​A:203–246 1plq​ :127–254 1plr​ :127–254 1rwz​A:121–241 1rxm​A:121–241 1rxz​A:121–241 1u76​C:127–254 1u7b​A:127–254 1ud9​C:200–243 1ul1​A:127–254 1vyj​G:127–254 1vym​C:127–254 1w60​B:127–254

Caudoviral

DNA polymerase accessory protein 45
Crystallographic structure of the trimeric gp45 sliding clamp from bacteriophage T4.[20]
Identifiers
Organism	Enterobacteria phage T4
Symbol	gp45
Entrez	1258821
PDB	1CZD
RefSeq (Prot)	NP_049666
UniProt	P04525
Other data
EC number	2.7.7.7
Chromosome	1: 0.03 - 0.03 Mb
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The viral gp45 sliding clamp subunit protein contains two domains. Each domain consists of two alpha helices and two beta sheets – the fold is duplicated and has internal pseudo two-fold symmetry.^[21] Three gp45 molecules are tightly associated to form a closed ring encircling duplex DNA.

Gp45 sliding clamp, N-terminal Identifiers Symbol DNA_pol_proc_fac Pfam PF02916 InterPro IPR004190 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB 1b77​ 1b8h​ 1czd​

Gp45 sliding clamp, C-terminal Identifiers Symbol Gp45_slide_clamp_C Pfam PF09116 InterPro IPR015200 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary PDB 1b77​ 1b8h​ 1czd​

Herpesviral

Some members of Herpesviridae encode a protein that has a DNA clamp fold but does not associate into a ring clamp. The two-domain protein does, however, associate with the viral DNA polymerase and also acts to increase processivity.^[22] As it does not form a ring, it does not need a clamp loader to be attached to DNA.^[23]

DNA polymerase processivity factor (HSV UL42, Alphaherpesvirus) Identifiers Symbol Herpes_UL42 Pfam PF02282 InterPro IPR003202 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Herpesvirus polymerase accessory protein (Betaherpesvirus) Identifiers Symbol Herpes_PAP Pfam PF03325 InterPro IPR004997 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Herpes DNA replication accessory factor (Gammaherpesvirus) Identifiers Symbol Herpes_DNAp_acc Pfam PF04929 InterPro IPR007013 Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Assembly

Sliding clamps are loaded onto their associated DNA template strands by specialized proteins known as "sliding clamp loaders", which also disassemble the clamps after replication has completed. The binding sites for these initiator proteins overlap with the binding sites for the DNA polymerase, so the clamp cannot simultaneously associate with a clamp loader and with a polymerase. Thus the clamp will not be actively disassembled while the polymerase remains bound. DNA clamps also associate with other factors involved in DNA and genome homeostasis, such as nucleosome assembly factors, Okazaki fragment ligases, and DNA repair proteins. All of these proteins also share a binding site on the DNA clamp that overlaps with the clamp loader site, ensuring that the clamp will not be removed while any enzyme is still working on the DNA. The activity of the clamp loader requires ATP hydrolysis to "close" the clamp around the DNA.